Thermal inactivation of Listeria monocytogenes during rapid and slow heating in sous vide cooked beef

T.B. HANSEN AND S. KNØCHEL. 1996. Heating at slowly rising temperatures is suspected to enhance thermotolerance in Listeria monocytogenes and, since anaerobic environments have been shown to facilitate resuscitation of heat-injured cells of this micro-organism, concern may arise about the possibility of L. monocytogenes surviving in minimally preserved products. The effect of rapid (> 10°C min^-1) and slow (0.3 and 0.6°C min^-1) heating on survival of L. monocytogenes in sous vide cooked beef was therefore examined at mild processing temperatures of 56, 60 and 64°C. No statistically significant difference ( P = 0.70) was observed between the tested heating regimes. Since the average pH of beef was low (5.6), and little or no effect was observed, a pH-dependency of heat shock-induced thermotolerance in L. monocytogenes is suggested to account for this result.     

Oxygen uptake and heart rate in young Green turtles (Chelonia mydas)

Oxygen uptake. heart rate and breathing frequencies were monitored in yearling Green turtles. Routine fed animals used about 100μ O₂ g live turtle^-1 h^-1 at 25C; this value was not significantly affected by size or short term food deprivation. Starved turtle showed a doubling of oxygen uptake after a satiation meal and heightened uptake persisted for five days. Between 15 and 30C oxygen consumption increased with rising temperature; below 15C there was falling temperature. Vigorous activity increased oxygen uptake to two or three times thr routine fed levels.
Turtles swimming gently at 25C exhibited a heart rate of around 46–48 beats min^-1; this rose to 64–68 beats min^-1 during vigorous and continuous activity. Contrary to expectations profound bradycardia was not seen during diving; even during 10 min dives a rate of 25–28 beats min^-1 was sustained. Significantly lower heart rates were only seen in turtles which were apparently asleep.     

Effect of hunger on the daily pattern of feeding rates in juvenile pink salmon, Oncorhynchus gorbuscha Walbaum

Juvenile pink salmon, Oncorhynchus gorbuscha , in two test groups of differing hunger level fed continuously on live copepods, maintained at high densities, throughout 12 h feeding periods in the laboratory. Analysis of video films showed that mean feeding rates were initially (first 10 min) between 51.5 and 63.8 prey-capture attempts (snaps) fish^-1 10 min^-1, but declined abruptly with increasing satiation to relatively constant levels of about 5.0 and 8.0 snaps fish^-1 10 min^-1, which were maintained during the subsequent 11 h of feeding. The data suggests that after the initial filling of their stomachs with food, juvenile pink salmon keep their stomachs full by feeding at a rate that balances the gastric evacuation rate of 18.6 mg prey h^-1 at 11° C. Less than 15% of the fish's stomach contents need be evacuated apparently for spontaneous feeding to resume or occur. Increased hunger level (by increasing a pre-test food deprivation period from 24 to 72 h) resulted in fish increasing their average feeding rate and thus their ration consumed from 23.6 to 39.8% dry body weight per 12 h.     

The effect of osmotic shock and subsequent adaptation on the thermotolerance and cell morphology of Listeria monocytogenes

The relationship between the time of exposure to different levels of NaCl and the corresponding changes in thermotolerance and cell morphology of Listeria monocytogenes was investigated. The kinetics of the increase in thermotolerance, after an osmotic upshift, showed a very rapid initial response (<2 min) followed by a more gradual increase whereby cells, after 4 h exposure at 30°C, became nearly as heat resistant as those grown for 48 h under the same conditions. Cells grown in media with 0.09 mol l⁻¹ NaCl subjected to a short osmotic up-shock in media containing 0.5, 1.0 or 1.5 mol l⁻¹ NaCl showed a 1.3, 2.5 and 8-fold increase in thermotolerance, respectively. Osmotic adaptation, signified by growth at the higher NaCl concentration, however, resulted in a 2- to 3-fold additional increase in thermotolerance. An osmotic down-shock caused a very rapid loss of thermotolerance (<5 min). Osmotic shock and adaptation experiments were also performed in minced beef where similar changes in thermotolerance were observed. Cell morphology was markedly affected by the osmolarity of the growth medium. Cells grown in media containing 1.5 mol l⁻¹ NaCl became up to 50 times longer than cells grown in media with 0.09 mol l⁻¹ NaCl, but no direct link to thermotolerance could be made.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

Purification and characterization of glutathione reductase from corn mesophyll chloroplasts

Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)^-1 min^-1. The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s^-1. The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s^-1.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

Stream channel experiments on downstream movement of recently emerged trout, Salmo trutta L. and salmon, S. salar L.—I. Effect of four different water velocity treatments upon dispersal rate

Four experimental stream channels were used to study instantaneous downstream dispersal rates of young trout, Salmo trutta L., and salmon, S. salur L ., relative to four different water velocities.
Young salmon showed a high rate of dispersal at a low velocity of 7.5 cm s⁻¹ and lower rates at higher velocities of 25 to 70cm s⁻¹. Trout showed their lowest rate at 25cm s⁻¹ with a slightly higher rate at 7.5 cm s⁻¹ and increasingly higher rates at velocities in excess of 25 cm s⁻¹. These results are consistent with field observations on the velocity preferences of young trout and salmon.     

Thermal inactivation of Listeria monocytogenes during a process simulating temperatures achieved during microwave heating

Conventional heating was used to expose cells of Listeria monocytogenes , either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10⁶ upon reaching 70°C. Simulated microwave cooking of L. monocytogenes in situ , on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85°C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10⁶. To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70°C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10⁶ and 10⁸. These results show that when a temperature of 70°C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes . The survival of this organism during microwave heating when temperatures of over 70°C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.     

Consumption, growth and evacuation in the Pacific cod, Gadus macrocephalus

Growth of Pacific cod was related to energy consumption (cal g⁻¹ day⁻¹) and was well described by linear equations. Maintenance ration was 11 and 12 cal g⁻¹ day⁻¹ at 4.5 and 6.5° C, respectively. Cod between 200 and 5000 g had similar growth rates when growth was expressed as a function of consumption (cal g⁻¹ day⁻¹). Laboratory consumption of food averaged 0.9 and 1.3% body weight per day at 4.5 and 6.5° C, respectively. At these temperatures growth was 0.34–0.38% body weight day⁻¹.
Maximum stomach volumes equated to approximately 4.7% of body weight with shrimp as prey. At this meal size Pacific cod did not feed the next day. A multiple meal evacuation experiment was used to verify the consumption estimates. A return-to-hunger estimate of the meal size evacuated was 1.5% body weight day⁻¹ at 6.5° C, similar to the 1.3% consumption estimate. For Pacific cod fed a single meal of 1% body weight the estimated instantaneous evacuation rate was 0.63 body weight day⁻¹ at 6.5° C. Meal size markedly affected the evacuation rate.
Measured consumption and growth rates are similar to those of Atlantic cod, Gadus morhua .     

Large-scale purification procedure of spinach leaf mitochondria —isolation and immunological studies of the F1–ATPase

A large–scale purification procedure for mitochondria from spinach ( Spinacia oteracea L, cv Medania) leaves is described. It involves differential centrifugation and density gradient centrifugation on a self–generating gradient of Percoll, From 3 kg of spinach leaves, 150 mg mitochondrial protein are obtained. The thylakoid contamination is lower than 0.2% on a chlorophyll basis. The mitochondria oxidize malate and glycine with state 3 rates of 108 and 140 nmol (mg protein)^-1 min^-1, with respiratory control ratios of 2,7 and 3,8 and ADP/O ratios of 2,0 and 2.1, respectively. The present large–scale purification procedure will facilitate further biochemical and molecular biological studies of leaf mitochondrial proteins.
A pure and active catalytic moiety of the F₁–ATPase (EC 3,6,1,3) was purified from the isolated mitochondria. The yield was 5 mg of F₁–ATPase from 150 mg mitochondria. The F₁–ATPase contained five polypeptides of apparent molecular mass 54 kDa (α), 52 kDa (β), 33 kDa (γ), 22 kDa (ω) and 11 kDa (ɛ). An additional component at 24 kDa was present in variable amounts in some preparations and was therefore not ascribed to the ATPase complex. The enzyme catalyzed ATP hydrolysis at a rate of 12.5 nmol (mg protein)^-1 min^-1. Antibodies against the spinach mitochondrial F₁–ATPase cross–reacted only with the a and β subunits of F₁–ATPases of spinach chloroplasts, photosynthetic bacteria Rhodospirillum rubrum and beef heart mitochondria.     

Destruction of phagocytosis-suppressing activity of aflatoxin B1 by ozone

The impact of ozone on the immunity-impairing activity of aflatoxin B₁ (AFB₁) was studied. Phagocytosis by rat peritoneal macrophages, which was found to be suppressed in the presence of AFB₁, remained unimpaired when the applied AFB₁ was pretreated with ozone (1.2 mg 1^-1) for 6 min at a flow rate of 40 ml min^-1. Hence, application of ozone on AFB₁-contaminated foodcrops seems to be a promising preventive measure against any adverse immunological disorder in consumers.     

Thermophilic glucoamylase from Talaromyces flavus

The heat-resistant mold, Talaromyces flavus , was found to produce a thermophilic glucoamylase that exhibited the highest activity at 50°C and in the pH range of 4.0–4.8. The K _m and V _max values of the crude enzyme for amylopectin were 0.21% and 16.7 mg glucose 1^-1, min^-1, respectively. The molecular weight of the enzyme as estimated by the gel filtration method was 42 kDa.     

Cardiorespiratory status of triploid brown trout during swimming at two acclimation temperatures

At 14° C, standard metabolic rate (75·1 mg O₂ h⁻¹ kg⁻¹), routine metabolic rate (108.8 mg O₂ h⁻¹ kg⁻¹), active metabolic rate ( c . 380 mg O₂ h⁻¹ kg⁻¹), critical swimming speed (U_crit 1·7 BL s⁻¹), heart rate 47 min⁻¹), dorsal aortic pressure (3·2 kPa) and ventilation frequency (63 min⁻¹) for triploid brown trout Salmo trutta were within the ranges reported for diploid brown trout and other salmonids at the same temperature. During prolonged swimming ( c . 80% U _crit), cardiac output increased by 2·3-fold due to increases in heart rate (1·8-fold) and stroke volume (1·2-fold). At 18° C, although standard and routine metabolic rates, as well as resting heart rate and ventilation frequency increased significantly, active metabolic rate and certain cardiorespiratory variables during exercise did not differ from those values for fish acclimated to 14° C. As a result, factorial metabolic scope was reduced (2·93-fold at 18° C v . 5·13-fold at 14° C). Therefore, it is concluded that cardiorespiratory performance in triploid brown trout was not unusual at 18° C, but that reduced factorial metabolic scope may be a contributing factor to the mortality observed in triploid brown trout at temperatures near 18° C.     

The effects of medium and rate of freezing on the survival of chlamydias after lyophilization

The effects of suspension media and rate of freezing on the survival of Chlamydia trachomatis LGV₂ and Chlamydia pneumoniae after lyophilization were assessed. The highest loss in infectious elementary bodies (EBs) occurred during lyophilization. The survival was higher after freezing at a rate of 1°C min^-1 and lyophilization than that after rapid freezing at - 70°C or - 196°C. The recovery (± 5%) was higher when fetal calf serum (FCS) containing glucose, saccharose or lactose were used as lyophilization media than that (0.5–3%) when yolk-sac, skimmed milk or phosphate buffer containing sucrose, glutamine and 10% FCS (SPG) were used. After lyophilization, the survival was not affected in the tested range from 10⁴ to 5 times 10⁶ inclusion-forming units (ifu) ml^-1 prior to freezing. After storage for 4 months at 4°C, the numbers of ifu of both Chlamydia serovars that were recovered were identical to the numbers of ifu immediately after lyophilization. It was concluded that chlamydias can be stored and transported in lyophilized form. However, a loss of 95% in infectious EBs should be taken into account.     

Purification and characterization of diacetyl reductase from Kluyveromyces marxianus

Diacetyl reductase from Kluyveromyces marxianus NRRL Y-1196 was purified 27.5-fold with a yield of 13% by ammonium sulphate fractionation, DEAE-anion exchange chromatography, hydroxyapatite chromatography and chromatofocusing. The purified enzyme was most active at pH 7.0 and exhibited optimal activity at 40°C. The K _m and V _max values for diacetyl were 2.5 mmol 1^-1 and 0.026 mmol 1^-1 min^-1, respectively. The enzyme did not react with monoaldehydes or monoketones, but reduced acetoin, diacetyl and methylglyoxal with NADH as a cofactor. The enzyme had an isoelectric point (pl) of pH 5.8, and its molecular weight was 50 kDa.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.     

Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive

The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10³ cfu g^-1 than on leaves inoculated with about 10⁵ cfu g^-1, but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested.     

Food intake and growth in the Moses perch, Lutjanus russelli (Bleeker), with reference to predation on penaeid prawns

Juvenile Moses perch were fed various rations of penaeid prawn and pilchard foods at 26.4 and 28.7° C to elucidate feeding–growth relationships. Maintenance rations amounted to 5.0 and 3.8% of body weight per day (BW day^–1) on prawn and pilchard diets, respectively, at 26.4° C and 4.5% BW day ¹ for prawn at 28.7° C. This apparent reduction in maintenance ration at higher temperature is probably due to reduction in activity levels. Starvation weight loss, an indicator of resting metabolic rate, increased with temperature and exhibited a Q¹⁰ of 2.0 to 2.4.
Fish fed ad libitum once or twice per day at 26.4° C grew at 1.1 % BW day^–1 and ingested about 9% of their BW day^–1. At 28.7° C fish ingested about 8% of their BW day^–1 and grew 1 .0% BW day^–1. These growth rates are comparable to those of similar sized lutjanids in the wild. Since prawns comprise 64% of the diet of Moses perch in northern Australia, this level of consumption would require about 6% BW day^–1 of prawns. The relevance of these findings to the assessment of predator impact on prawns in Australian estuaries is discussed.     

Protoplasts from pectinolytic fungi: isolation, regeneration and pectinolytic enzyme production

S. Solís, M.E. FLORES AND C. HUITRON. 1996. Protoplast release in pectinolytic strain mutants of Aspergillus sp. CH-Y-1043 (A13) and Aspergillus flavipes ATCC-16795 (F7) is described. Optimum yield of protoplasts A13 was obtained in a lapse of 1 h when commercially lytic enzymes of Trichoderma harzanium (2 mg ml⁻¹) were added in 0.05 mol 1⁻¹ citrate-phosphate buffer pH 5.0 containing 0.7 mol 1⁻¹ KCl and 10 mg ml⁻¹ BSA. Best results in F7 were obtained when the protoplasting system of A13 was supplemented with 10 mg ml⁻¹ Aureobasidium sp. lytic enzymes. Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 0.7 mol 1⁻¹ KCl and sorbitol were used as osmotic stabilizers. Endo-P, Exo-P and pectin lyase production were not modified during the process of regeneration.