The presence of endotoxin in powdered infant formula milk and the influence of endotoxin and Enterobacter sakazakii on bacterial translocation in the infant rat

Lipopolysaccharide (LPS) is a heat stable endotoxin that persists during the processing of powdered infant formula milk (IFM). Upon ingestion it may increase the permeability of the neonatal intestinal epithelium and consequently bacterial translocation from the gut. To determine the level of endotoxin present in IFM, 75 samples were collected from seven countries (representing 31 brands) and analysed for endotoxin using the kinetic colorimetric Limulus amoebocyte lysate (LAL) assay. The endotoxin levels ranged from 40 to 5.5 x 10(4) endotoxin units (EU) per gram and did not correlate with the number of viable bacteria. The neonate rat model was used to address the risk of endotoxin-induced bacterial translocation from the gut. Purified Escherichia coli LPS was administered to rat pups followed by inoculation with Enterobacter sakazakii ATCC 12868. Bacteria were isolated from the mesentery, spleen, blood and cerebral spinal fluid (CSF) of endotoxin-treated rats due to enhanced gut and blood brain barrier penetration. Histological analysis of the colon showed marked distension of the mucosal and muscular layers. It is plausible that the risk of neonatal bacteraemia and endotoxemia, especially in neonates with immature innate immune systems, may be raised due to ingestion of IFM with high endotoxin levels.     

Caseinolytic and milk-clotting activities from Moringa oleifera flowers

This work reports the detection and characterization of caseinolytic and milk-clotting activities from Moringa oleifera flowers. Proteins extracted from flowers were precipitated with 60% ammonium sulphate. Caseinolytic activity of the precipitated protein fraction (PP) was assessed using azocasein, as well as α(s)-, β- and κ-caseins as substrates. Milk-clotting activity was analysed using skim milk. The effects of heating (30-100°C) and pH (3.0-11.0) on enzyme activities were determined. Highest caseinolytic activity on azocasein was detected after previous incubation of PP at pH 4.0 and after heating at 50°C. Milk-clotting activity, detected only in the presence of CaCl(2), was highest at incubation of PP at pH 3.0 and remained stable up to 50°C. The pre-treatment of milk at 70°C resulted in highest clotting activity. Enzyme assays in presence of protease inhibitors indicated the presence of aspartic, cysteine, serine and metallo proteases. Aspartic proteases appear to be the main enzymes involved in milk-clotting activity. PP promoted extensive cleavage of κ-casein and low level of α(s)- and β-caseins hydrolysis. The milk-clotting activity indicates the application of M. oleifera flowers in dairy industry.     

Influence of lactate and acetate salt adaptation on Salmonella Typhimurium acid and heat resistance

The aim of the present study was to determine the survival of Salmonella Typhimurium adapted with sodium lactate (NaL), potassium lactate/sodium acetate mixture (KL/NaA) or sodium acetate (NaA) in simulated gastric fluid (SGF) and during heat treatment. NaL-, KL/NaA- and NaA-adapted cells were prepared by incubating in tryptic soy broth (TSB) containing these salts at 5, 5 and 3% (w/v) concentration levels, respectively, for 24 h at 37 °C. The Baranyi model was used to compare the growth kinetic parameters of adapted cells. The acid and heat resistance of adapted cells were determined by incubating in SGF (pH 2.04) at 37 °C and in TSB at 55.8, 57.8 and 59.8 °C, respectively. Adapted cells had significantly (P < 0.05) longer lag phase duration (LPD) and slower maximum growth rate (MGR) than non-adapted cells. The acid resistance of KL/NaA-adapted cells was not significantly (P > 0.05) different from that of non-adapted cells. NaL-adapted cells were more susceptible to the low pH environment, whereas NaA-adapted cells showed enhanced acid resistance compared to non-adapted and other adapted cells. Unlike acid resistance, both NaL- and NaA-adapted cells showed enhanced heat resistance with increased D-values, regardless of treatment temperatures. Thus, this study indicates that adaptation of S. Typhimurium to 5% NaL or 3% NaA could enhance their ability to survive thermal processes or in the human stomach, possibly increasing the risk of Salmonella outbreaks.     

Complete detoxification of tris(2-chloroethyl) phosphate by two bacterial strains: Sphingobium sp. strain TCM1 and Xanthobacter autotrophicus strain GJ10

Tris(2-chloroethyl) phosphate (TCEP), a flame retardant, is recently regarded as a potentially toxic and persistent environmental contaminant. We previously isolated TCEP-degrading bacterium, Sphingobium sp. strain TCM1, which, however, produced a toxic metabolite: 2-chloroethanol (2-CE). This study was undertaken to develop a detoxification technique of TCEP using strain TCM1 with a 2-CE-degrading bacterium: Xanthobacter autotrophicus strain GJ10. TCEP degradation by strain TCM1-resting cells was thermally stable for 30 min at 30 °C. It was optimal at 30 °C and at pH 8.5. In the optimum condition, TCM1 cells up to a final cell density of 0.8 at OD(660) in the reaction mixture were unable to hydrolyze the phosphotriester bonds of 10 μM TCEP completely. The addition of 50 μM Co(2+) to reaction mixture enhanced the hydrolysis and caused the complete hydrolysis at the cell density of 0.8. Strain GJ10 resting cells degraded 2-CE only slightly, which might be attributable to lack of coenzyme regeneration of enzymes involved in the degradation. In contrast, the growing cells degraded approximately 180 μM of 2-CE within 24 h. Based on these results, we designed a two-step TCEP detoxification reaction consisting of TCEP hydrolysis to 2-CE by strain TCM1-resting cells and the following degradation of the resulting 2-CE by strain GJ10-growing cells. The combined reaction completely detoxified 10 μM TCEP, and thus opens a way to microbial detoxification of the potential toxic, persistent organophosphorus compound.     

The effect of heat shrink treatment and storage temperature on the time of onset of "blown pack" spoilage

This study determined the effects of (a) the short "heat shrink" treatment frequently applied to vacuum packed meats within normal commercial production, and (b) chill holding storage temperature, on the subsequent time to onset (TTO) of "blown pack" spoilage (BPS). Beef or lamb steaks were inoculated with 103 CFU/cm2 of spore suspensions of five gas producing clostridia, vacuum packed (VP) and treated as follows: no heat, 50°C/15 s, 70°C/10 s or 90°C/3 s. Samples were stored at -1.5, 1 or 4°C and examined daily to determine TTO of BPS. For each strain, pack treatment and storage temperature had significant (P<0.05 and P<0.001 respectively) effects on TTO of BPS, i.e. 90°C/3 s<70°C/10 s<50°C/15 s≤"no heat", and 4°C<1°C<-1.5°C. The study suggested that the meat industry could reduce the risks of BPS by avoiding higher temperature (90°C/3 s or 70°C/10 s) heat shrinking, and by storing VP meats at lower temperatures (e.g. -1.5°C).     

Effect of high pressure on the regulation of phosphorylase activity in pre-rigor rabbit muscle

The effects of high pressure (150 MPa) on the regulation of phosphorylase activity in pre-rigor rabbit muscles have been studied at 35° and 0°C. At 35°C muscle contracts, phosphorylase is activated and the muscle pH falls to 5·8 in 2 min. Coinciding with these changes, phosphorylase phosphatase activity falls rapidly, while phosphorylase kinase, although active for longer, loses its activity as the pH falls. Both of these enzymes are completely inactivated after 5 min under pressure, while phosphorylase still retains 80% of its activity under these conditions. The effects of pressure on the activities of these enzymes in white and red muscles of rabbits were compared, with a greater effect being observed in white muscles. At 0°C, muscles subjected to high pressure did not contract, but at this temperature the three enzyme activities (phosphorylase, phosphorylase kinase and phosphorylase phosphatase) were all lost at a greater rate than at 35°C, although the pH of the muscle did not fall below 6·5. The effects of high pressure treatment on isolated phosphorylase a and b and phosphorylase kinase were also studied at both 0° and 35°C and the results obtained closely paralleled those observed in whole muscle.     

Development of a heat-processing method for koji to enhance its antioxidant activity

We developed a heat-processing method to enhance the antioxidant activity of koji. The superoxide anion scavenging activity (SOSA) and oxygen radical absorbance capacity (ORAC) of heat-processed koji (HP-koji) at 55 °C for 7 days were 4.9 times and 4.2 times, respectively, those of unheated koji. These results showed that heat processing effectively enhances the antioxidant activity of koji. Analysis of the antioxidant activities of koji subjected to a range of temperatures (45-75 °C) revealed that the SOSA is enhanced by heating at higher temperatures, which might be catalyzed by Maillard reaction, whereas the ORAC was enhanced by heating at lower temperatures, which might be catalyzed by an enzymatic reaction. Assuming these enhancements in antioxidant activities are contributed by both Maillard and enzyme reactions, we hypothesized that the antioxidant activity of HP-koji could be more effectively amplified by heating at a higher temperature after the progression of the enzymatic reaction at a moderate temperature. Therefore, we evaluated the effect of heating of koji in a stepwise manner, first at 55 °C for 2 days and then at 75 °C for 5days. The antioxidant activities of stepwise-heated HP-koji were higher than those of koji heated at either 55 °C or 75 °C. The SOSA and ORAC of stepwise-heated HP-koji were 94 times and 6 times, respectively, those of unheated koji. This result suggests that enzymatic reaction followed by Maillard reaction can effectively enhance the antioxidant activity of HP-koji. Thus, we developed a novel heat-processing method to enhance the antioxidant activity of koji.     

Formation of lysinoalanine during heat treatment of milk

The heat-induced formation of lysinoalanine (LAL) was studied in raw skim milk that had been subjected to heat treatments as is usual in milk technology. Preheat treatment of milk at temperatures below 100 degrees C up to 20 min resulted in neglectable LAL amounts below 10 ppm i.p. (i.p.=in the protein) if at all. Tests with an UHT pilot plant showed that there was no proven formation of LAL with direct UHT-treatment at 110-130 degrees C for 10-25 min. In indirect UHT-treated milk vert small LAL amouints up to 50 ppm i.p. were detected only in those milk samples that were treated at temperatures high than 145 degrees C and holding times longer than 10 s. Autoclave sterilization in the range of 110-129 degrees C/10-25 min induced LAL amounts of 110 to 710 ppm i.p.. LAL formation in autoclave sterilized milk was almost directly dependent on heating temperature and time. Pre-treatment at temperatures below 100 degrees C induced no further LAL formation in any sterilization processes subsequent to preheating. In the pH range 6,50-6,90 LAL amounts in autoclave-sterilized milk increased directly with higher pH values at all temperatures. The varying degree of LAL formation with pH value was substantially more noticeable at higher than at lower temperatures. Increasing of lactose concentration caused only an insignificant decrease in LAL formation in autoclave-sterilized milk.     

Photodegradation of rhodamine B in water assisted by titania nanorod thin films subjected to various thermal treatments

Well-crystallized titania nanorod thin films with high specific surface areas were synthesized through in situ oxidation of metallic titanium substrates with hydrogen peroxide solutions at a low temperature of 80 degrees C. The effects of thermal treatments on the ability of the thin films to assist photodegradation of rhodamine B in water were studied in detail. It is found that, due to a unique nanofeature, the titania nanorod film retained its small grain size and high specific surface area during the subsequent thermal treatment for up to 450 degrees C, which further improved the crystallinity of titania. In addition, upon heating to 450 degrees C, the nanorods still possessed a high surface fractal dimension, which is an indication of high surface roughness and surface area. As a result, the photocatalytic activity of the titania nanorod film is found to increase with an increasing heating temperature for up to 450 degrees C. The subsequent thermal treatment at temperatures beyond 450 degrees C decreased the photocatalytic activity, because of the significantly reduced specific surface area. The current investigation provided a simple and easily scaled-up approach to produce photocatalysts for efficient removal of dye effluents in wastewater.     

Rheological changes during isothermal holding of salted beef homogenates

The gelation of lean meat homogenates was examined in a thermal scanning rheology monitor, a device that non-destructively measures rigidity and elasticity, as response to a repetitive small movement. Salted (NaCl and pyrophosphate) homogenates were scanned from 10°C to 85°C at 1°C/min, continuously or with an isothermal hold period at 25, 35, 45, 50 or 55°C. The intention of isothermal holding was to visualize key features of the gelation process. Whatever the thermal path, the final properties of the gel were substantially the same. This indicates that heat-mediated gelation follows only one path and is complete by 85°C. The hold at 55°C yielded the most information on gelation. During the hold, gelation was very nearly complete while rigidity steadily increased. This showed that transition from the sol state to the gel and rigidity development are separate events. Samples held at 25°C underwent pH-dependent transitions in rheological properties but at higher hold temperatures these transitions were often not observed. They were caused by a progressive loss of pyrophosphate due to an endogenous pyrophosphatase. The pH and temperature dependencies of the transitions were easily explained by classic enzymology. The enzyme activity could be inhibited by 10 mm NaF. Under these conditions the onset of final gelation occurred at a slightly, but significantly, lower temperature. Models to explain this and another subtle effect of pyrophosphate are presented. At a given pH, although gelation is subtly affected by pyrophosphate, the final rigidity and elasticity are much the same in its presence or absence. Pyrophosphate is in near universal use in salted meat products. Presumably its reported advantages, better water-holding capacity and greater gel strength, are not revealed by the thermal scanning technique.     

Effect of low voltage electrical stimulation and temperature conditioning on postmortem changes in glycolysis and calpains activities of Korean native cattle (Hanwoo)

The combined effects of low voltage electrical stimulation (LVES) and temperature conditioning during early postmortem (PM) ageing on glycolytic rates and calpains activities of Korean native cattle (Hanwoo) were determined. M. longissimus was taken after splitting course, divided into three pieces and temperature conditioned at 2, 16, and 30°C for 3 h PM. The PM glycolytic rates, calpains and calpastatin activities were measured at 1, 3, 9, and 24 h PM. Although both LVES and the 30°C treatment accelerated glycolytic rates and resulted in improved enzyme activities, LVES was more effective for the improvement of enzyme activities than the 30°C treatment. Among tested Rigor-values (R-value; R(248), R(250), and R(258)), R(258) showed the highest correlation with pH (r=0.814, P<0.01), glycogen content (r=0.784, P<0.01) and μ-calpain (r=0.838, P<0.01) and selected as a suitable parameter to predict glycolytic rate. The high correlation coefficients between μ-calpain activity and metabolic rate parameters suggest that the change in the enzyme activity is closely related to glycolytic rates. The LVES in combination with the 30°C treatment until 3 h PM was the best treatment to accelerate the glycolytic rate and to improve the calpains activities in Hanwoo tissue.     

In situ inactivation of polyphenol oxidase in mamey fruit (Pouteria sapota) by microwave treatment

Polyphenol oxidase (PPO) is the enzyme responsible for quality loss in most fruits and vegetables. Quality loss is mainly because of oxidative chemical reactions which generate the darkening of tissues. Mamey fruit (Pouteria sapota) after harvesting suffers a rapid quality decay trough activation of PPO. However, PPO may be inactivated in situ by chemical or thermal treatment. In food processing, microwave treatment (MT) has been used recently as an alternative for PPO inactivation. In this study, it was observed that mamey fruit pulp subjected to a gently MT resulted in a higher PPO activity as the generated heat induced in situ the increase in PPO activity. In contrast, PPO was completely inactivated after long MT by using a high microwave power. Temperature in mamey pulp after MT reached a maximum of 79 °C; although PPO was active up to 60 °C. PPO was completely inactivated when conventional blanching treatment was performed but required a higher temperature (92 °C/300 s). The optimum energy intensity (E(opt)) for PPO inactivation by MT was 0.51 kJ/g or 937 W/165 s. Under this condition, the remaining PPO activity was inversely proportional to energy intensity (E). Interestingly, MT resulted in a negligible damage in microstructure of mamey pulp, although blanching treatment resulted in large damaging effects on tissue organization and shape. Therefore, MT is proposed as an effective way to completely inactivate PPO without causing any significant damage to fruit tissues and shape; as preservation of color, flavor, and taste would be favored.     

Thermophysical characterization of tilapia myosin and its subfragments

Purified tilapia myosin was digested with α-chymotrypsin and purified to obtain heavy meromyosin (HMM) and light meromyosin (LMM). The thermophysical properties of Tilapia myosin, HMM, and LMM were characterized. Constantly heated myosin, HMM, and LMM samples showed that aggregates began to form around 40 °C as evidenced by the increase of turbidity for all 3 samples (0.25 mg/mL). Beginning turbidity measurements showed differing levels of absorbance for each protein fragment with increasing absorbance values in the following order HMM, myosin, and LMM (0.0026, 0.0282, and 0.052, respectively). Differential scanning calorimetry showed 3 (17.5, 41.9, and 49.9 °C), 2 (43 and 67.1 °C), and 3 (40.4, 51.7, and 69 °C) major peaks for myosin, HMM, and LMM, respectively. Dynamic rheology measurements demonstrated crossover points, which are generally recognized as gelation point, 40.3 °C for myosin and 27 °C for HMM. The results shown for the thermally stable properties of tilapia myosin, HMM, and LMM showed clear evidence that they are all thermal stable at temperatures ranging from 10 °C to approximately 40 °C after which they all are completely denatured. The results also showed that the thermo stability of the myosin and its subfragments were greatly influenced by fish habitat temperature.     

Effect of a bacteriocin produced by Pediococcus acidilactici against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat

The inhibitory effect of a bacteriocin, produced by Pediococcus acidilactici, against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat surface, was evaluated by in situ assays. Samples were incubated with the bacteriocin and then with a culture of the pathogenic bacteria. The treatment with 500, 1000 or 5000 bacteriocin units/ml (BU/ml) reduced the counts of L. monocytogenes after storage at 15°C during 72h by 1, 2 or 3 log cycles and with 1000 or 5000 BU/ml after storage at 4°C during 21 days by 2.5 or 3.5 log cycles, respectively, compared to the control. With C. perfringens a bacteriostatic effect could be observed.     

Effect of high pressure, temperature, and storage on the color of porcine longissimus dorsi

The color of pork longissimus dorsi high pressure (HP) treated at 200 to 800MPa at 5 and 20°C for 10min was determined to a high degree by pressure level and to a lesser degree by temperature. Severe color changes appeared up to a threshold pressure at 400MPa. HP treatment at 20°C compared to 5°C resulted in meat, which was less red and slightly lighter. Storage at 2°C for 6days had no effect on lightness due to no further protein denaturation, but meat HP treated above 300MPa became significantly less red and more yellow within the first day of storage. Reflectance spectra showed that a short-lived ferrohemochrome myoglobin species was formed during HP treatment at 300 to 800, but transformed into a brown, ferric form of the pigment within the first day of storage. This explains the observed changes in the redness and yellowness after one day of storage.     

Inactivation of Salmonella spp. in liquid whole egg using pulsed electric fields, heat, and additives

This paper evaluates the lethal effectiveness on 7 different Salmonella serovars of the application, in static and continuous conditions, of pulsed electric fields (PEF) followed by heat treatments in liquid whole egg (LWE) with additives (EDTA or triethyl citrate-TC-). Compared to heat treatments, the PEF (25 kV/cm and 75-100 kJ/kg) followed by heat (52°C/3.5', 55°C/2', or 60°C/1') in LWE with 2% TC permitted the reduction of heat treatment time from 92 fold at 52°C to 3.4 fold at 60°C, and 4.8 fold at 52°C in LWE with EDTA for a 9-Log(10) reduction of the population of Salmonella Enteritidis. The new designed treatments inactivated more than 5 Log(10) cycles of Salmonella serovars Dublin, Enteritidis 4300, Enteritidis 4396, Typhimurium, Typhi, Senftenberg, and Virchow, both in static and continuous conditions. Conversely, current heat pasteurization treatments of 60°C/3.5' and 64°C/2.5' reduced 5 Log(10) cycles of various serovars of Salmonella but only 2 and 3-4 Log(10) cycles of Salmonella Senftenberg and Salmonella Enteritidis 4396, respectively. Soluble protein content (SPC) decreased 1.8%, 1.3%, and 5.0% after the successive application of PEF followed by heat at 52, 55, and 60°C in the presence of 2% TC, respectively, whereas 1.6% and 9.4% of SPC were reduced after heat pasteurization at 60 and 64°C, respectively. Results indicate that designed treatments could be an alternative to current heat pasteurization of LWE as showed higher lethal effectiveness against Salmonella serovars with a similar or even lower decrement of the soluble protein content.     

Thermal inactivation of Escherichia coli O157:H7 and Salmonella on catfish and tilapia

Thermal inactivation kinetics of individual cocktails of Escherichia coli O157:H7, or of Salmonella meat isolates or seafood isolates were determined in catfish and tilapia. Determinations were done at 55, 60 and 65 °C using a circulating-water bath and calculated using linear regression analysis. Salmonella seafood and meat isolates D-10 values on the finfish were the same and ranged from 425 to 450, 27.1 to 51.4, 2.04-3.8 s (z = 4.3 °C) at 55, 60 and 65 °C, respectively. The E. coli O157:H7 D-10 values ranged from 422 to 564, 45.2 to 55.5 and 3.3-4.2 s (z = 4.3 °C) at 55, 60 and 65° C, respectively. The only statistical difference (P ≤ 0.05) was found when comparing the D-10 values for E. coli O157:H7 at 55 °C on catfish and tilapia. The other D-10 values for the Salmonella at all temperatures and E. coli O157:H7 at 60 and 65 °C on the catfish or tilapia showed no statistical difference. D-10 values for the catfish and tilapia were significantly lower than the reported values in other food systems, but the z-values were within the literature reported range. These D-10 values can be used to determine cooking parameters of finfish.     

Updating on the lysinoalanine content of commercial infant formulae and beicost products

Since the safety issue of lysinoalanine (LAL) still remains unresolved, its concentration in infant formulae should be reduced to a minimum. Data collected in the 1980s indicated that LAL is formed in higher amounts in liquid than in powdered formulae. Recently the market of liquid infant formulae is increasing rapidly and there are no new data, so 23 commercial powdered or liquid samples were investigated. In powdered samples, LAL was below the detection limit, whereas liquid adapted formulae contained up to 86 μg/g protein, liquid follow-on formulae up to 390 μg/g protein, and liquid growing milks up to 514 μg/g protein. The concentration of LAL in liquid formulae is considerably lower than in the past; however, the level in a few products remains rather high, especially compared with normal UHT-treated milk. Great differences were observed among products of different companies, which suggests that labelling with the thermal treatment applied would be very advisable. The investigation of some beicost products indicates that LAL is present only in products certainly containing milk proteins. Considering the rather low levels in comparison with liquid infant formulae, the contribution of beicost products to the total LAL daily intake does not seem to be particularly relevant.     

Effect of Heat and Alkali on Lysinoalanine Formation in Fish Muscle

Alkali-treated fish products such as lutefisk and sodium tripolyphosphate-treated fillets were analyzed for lysinoalanine (LAL). No detectable amounts of LAL were found in these products both of which had pH values between 8.0 and 8.5 even after they had been heated to ordinary cooking temperatures (162°C) for up to 30 min. There was no measurable quantity of LAL found in samples at pH 10 heated for 60 min at 90°C; however, measurable quantities of LAL were found in similarly heated samples at pH 12 and 13. Thus, LAL formation is not an apparent problem in the ordinary processing of fish.