Effects of mastic resin and its essential oil on the growth of proteolytic Clostridium botulinum

Studies were done to determine the effect of mastic resin and its essential oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of Clostridium botulinum in media, and on neurotoxin production in challenge studies with English-style crumpets. Preliminary studies, using a spot-on-the-lawn method, indicated that high levels of mastic resin in ethanol ( approximately 8% w/w) were required for complete inhibition of all strains of C. botulinum tested, but mastic resin in ethanol had a greater anti-botulinal effect than ethanol alone. However, only low levels of mastic oil ( approximately 0.3% v/v) were required for inhibition of proteolytic strains of C. botulinum. Both studies showed a strain specific inhibition, with C. botulinum type A strains being more sensitive to mastic resin and its essential oil than type B strains. However, mastic resin in ethanol proved to be more effective when used as a vapor phase inhibitor applied to cotton pads and placed inside inoculated plates than when added directly to media. While both mastic resin and its essential oil inhibited the growth of proteolytic strains of C. botulinum in vitro, they failed to inhibit neurotoxin production in challenge studies with C. botulinum in English-style crumpets.     

Growth and germination of proteolytic Clostridium botulinum in vegetable-based media

The growth of proteolytic Clostridium botulinum from spore inocula and changes in spore counts in mushroom, broccoli, and potato purées were monitored. Four strains of proteolytic C. botulinum types A and B were inoculated separately at approximately 10(4) spores per ml in nutrient broth and vegetable purées incubated at 15, 20, and 30 degrees C for up to 52 days. The times for the cell populations to increase 1,000-fold (T1,000) in the tested vegetables (1 to 5 days at 30 degrees C, 3 to 16 days at 20 degrees C, 7 to > 52 days at 15 degrees C) were similar to those for meat or fish. Only temperature significantly influenced growth rate. In contrast, the lag phase depended on the strains and media tested, in addition to temperature. Lag times and T1,000S for proteolytic C. botulinum were longer for potato and broccoli purées than for mushroom purée. These differences were not related to different pHs or redox potentials. The germination level, evaluated as the decrease in the spore count, was low. The addition of a germinant mixture (L-cysteine, L-alanine, and sodium lactate) to some strains inoculated in vegetable purées resulted in an increase in germination, suggesting a lack of germination-triggering agents in the vegetable purées.     

Influence of the conditions of storage and cooking on growth,invasion and survival of Salmonella enteritidis in eggs

Basal studies for the confirmation of sanitary rules in the kitchen were performed, focusing on preventing an outbreak of food poisoning due to eggs contaminated with Salmonella Enteritidis (SE), using hen and quail eggs. SE did not grow at 5 degrees C but grew markedly at 25 degrees C in eggs. The invasion and growth of SE were marked under very humid conditions regardless of whether the eggshell was damaged. The invasion of SE into egg also occurred when eggs were taken in and out of the refrigerator. Moreover, SE was spread immediately to all non-contaminated eggs when SE-contaminated eggs were cracked into a bowl with non-contaminated eggs. In homemade mayonnaise containing 15% vinegar, sterilization took several hours to occur. On a stainless-steel bowl, SE survived for 2 weeks or more. These findings suggest that it is necessary to pay attention to secondary contamination.     

Effect of ethanol on the growth of Clostridium botulinum

Model broth studies were carried out to investigate the effect of ethanol on the growth of proteolytic (group I) strains of Clostridium botulinum. Ethanol extended the pathogen's lag phase, decreased its exponential growth rate, and decreased its final level of growth in the stationary phase. In all cases, botulinum neurotoxin production was associated with growth. Micrographs of C. botulinum cultures grown at 37 degrees C in trypticase peptone glucose yeast extract (TPGY) broths containing 2 and 4% ethanol showed elongation of vegetative cells and interference with cell division. The inhibition of growth and toxin production at the ethanol level predicted (5.5%, wt/wt) was confirmed by microscopy and by the mouse bioassay. A subsequent study was carried out to determine the combined effect of ethanol (0 to 8% [wt/wt]), water activity (aw; 0.953 to 0.997), and pH (6.2 to 8.2) on the probability of the growth of and neurotoxin production by proteolytic strains of C. botulinum (10(3) spores per ml). Growth and neurotoxin production occurred in 1 to 3 days in TPGY broths without ethanol (0%) and in 2 to 4 days in broths containing 2% ethanol regardless of the aw or pH levels (P < 0.005). Growth and neurotoxin production were delayed by an ethanol concentration of 4% ethanol and completely inhibited by a concentration of 6%. At an ethanol concentration of 4%, the probability of growth and toxin production over 365 days (Pt) was influenced by aw and pH. After 365 days, the maximum probability of growth and toxin production (Pmax) was 1 for all but one combination. However, tau, the time it took for 50% of all eventually positive replicates for any given combination of barriers to show growth and/or turbidity, ranged from <3 to 229 days. All tubes of TPGY broths that showed no growth after 365 days were subcultured in fresh TPGY broths. In all cases, growth and toxin production occurred within 24 h at 37 degrees C, indicating the reversible (sporostatic and/or bacteriostatic) effect of ethanol on C. botulinum.     

Evaluation of survival of Staphylococcus aureus and Clostridium botulinum in charqui meats

Charqui meats were prepared in laboratory conditions in order to carry out experiments to observe the possibility of development of enterotoxigenic Staphylococcus aureus and Clostridium botulinum proteolytic type B spores and their toxins. Results demonstrated that the harsh processing conditions, high salt concentration, relative high temperature, a(w) values, inhibited the growth of both bacteria. Under our experimental conditions, S. aureus would survive throughout the sequence of salting steps i.e. brine followed by rock salting and the sunshine drying step. However, at final a(w) value of 0.70-0.75 would create conditions to inhibit its development. The other experiment revealed that C. botulinum spores germination also was impaired because of these low a(w) values. Under these conditions, charqui meats revealed to be safe products in relation to toxins from both enterotoxigenic S. aureus and C. botulinum.     

Estimating the survival of Clostridium botulinum spores during heat treatments

A recently published study of the inactivation of Clostridium botulinum spores at various temperatures in the range of 101 to 121 degrees C and neutral pH revealed that their semilogarithmic survival curves all had considerable upward concavity. This finding indicated that heat inactivation of the spores under these conditions did not follow a first-order kinetics and that meaningful D values could not be calculated. The individual survival curves could be described by the cumulative form of the Weibull distribution, i.e., by log S = -b(T)t(n(T)), where S is the survival ratio and b(T) and n(T) are temperature-dependent coefficients. The fact that at all temperatures in the above range n(T) was smaller than 1 suggested that as time increases sensitive members of the population parish and survivors with increasing resistance remain. If damage accumulation is not a main factor, and the inactivation is path independent, then survival curves under monotonously increasing temperature can be constructed using a relatively simple model, which can be used to calculate the spores' survival in a limiting case. This is demonstrated with computer-simulated heating curves and the experimental constants of the C. botulinum spores, setting the number of decades reduction to 8, 10, and 12 (the current criterion for commercial sterility).     

Influence of growth conditions on heat-stable phospholipase activity in Pseudomonas

Many psychrotrophic bacteria contaminating raw milk produce phospholipase that withstands pasteurization and UHT treatments. This enzyme acts on the milk fat globule membrane and exposes triacylglycerides to the action of lipase. Phospholipase production by various isolates of Pseudomonas was investigated. The isolates were cultured aerobically at 8 degrees C in nutrient broth, McKellar's minimal salts medium, Chrisope's medium, and skim milk. Each strain produced phospholipase during the 50 h incubation. Enzyme production varied significantly (P < 0.001) with strain and growth medium. Strains varied significantly (P < 0.001) in their enzyme production in each medium and during the incubation time as well. Strain, incubation time, and the growth medium significantly influenced (P < 0.001) heat stability of the enzyme activity. Pasteurization reduced the activity, but did not eliminate it in skim milk.     

Effects of storage temperature and water activity on the survival of bifidobacteria in powder form

Effects of water activity and storage temperature on survival of bifidobacteria in powder form were investigated and kinetic analyses were performed to reveal characteristics of the stability. A significant positive correlation was observed between water activity and natural logarithm of the inactivation rate constant of bifidobacteria powder, indicating that higher water activity induced lower stability of bifidobacteria in powder form. Also, higher temperature condition induced lower survival rate, which was supported by that the stability was followed the Arrhenius theory. These findings constructed a prediction model for bifidobacteria survival in powder form.     

Influence of storage conditions of apples on growth and patulin production by Penicillium expansum

Penicillium expansum causes blue mould rot, a serious post-harvest disease of apples, and is the main producer of the mycotoxin patulin. The present study aimed to determine the influence of storage conditions (i.e. temperature and O(2) level) on growth and patulin production by different P. expansum strains on a simulation medium and on apples. Growth was strongly influenced by the temperature, while the used atmosphere (20, 3, and 1% O(2); <1% CO(2)) had no effect. Optimal growth was observed at 25 degrees C for every strain tested. Patulin production was stimulated when the temperature decreased (from 20 to 10 or 4 degrees C), while a further decrease of the temperature to 1 degrees C caused a reduction in patulin production. The temperature at which the stimulation was changed into suppression was strain dependent. Similar results were observed for the O(2) level. A reduction of the O(2) level from 20 to 3% O(2) could stimulate or suppress patulin production depending on the strain, while a clear decrease of the patulin production was observed when the O(2) level was reduced from 3 to 1%. These results show that the induction of limited stress to the fungus, such as lowering the temperature or lowering the O(2) levels stimulates patulin production. However, the combination of different stress conditions (e.g. low temperature and low O(2)) will result in a reduced formation of the toxin. The combination of stress conditions, at which the transition from stimulation to suppression is observed, is strain dependent. Moreover, patulin production is characterized by a high natural variability. The presented results show that the temperature and O(2) level has to be as low as possible during the storage of apples in order to suppress patulin production and to guarantee food safety.     

Antiaflatoxigenic property of food grade antioxidants under different conditions of water activity in peanut grains

Analytical grade (AG) and industrial grade (IG) of three-food grade antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl paraben (PP) were analyzed to prove their fungitoxic effect on Aspergillus section Flavi strains. The effect of interactions among 10 antioxidant treatments at water activity levels (0.982, 0.955, 0.937 a(W)) for 11 and 35 days of incubation and at 25 degrees C in peanut grains on mycelial growth (CFU g(-1)) and aflatoxin B(1) (AFB(1)) accumulation were evaluated. Both antioxidant grade treatments had a significant effect (P<0.001) on fungal count. All antioxidant treatments showed the highest effectiveness on control of growth of peanut aflatoxigenic strains at 0.937 a(W) and at 11 days of incubation. Overall, AG and IG binary mixtures M3 (20+10 mM), M4 (20+20 mM) and ternary mixtures M5 (10+10+10 mM), M6 (10+20+10 mM), M7 (20+10+10 mM) and M8 (20+20+10 mM) were the treatments most effective at inhibiting growth of Aspergillus section Flavi strains. Industrial grade BHA 10 and 20 mM, binary mixtures M1 (10+10 mM), M2 (10+20 mM), M3 (20+10 mM), M4 (20+20 mM) and ternary mixtures M5 (10+10+10 mM), M6 (10+20+10 mM), M7 (20+10+10 mM) and M8 (20+20+10 mM) completely inhibited AFB(1) production. The studied results suggest that IG antioxidant mixtures have potential for controlling growth of these mycotoxigenic species and prevent aflatoxin accumulation at the peanut storage system.     

不同贮藏条件下花生中黄曲霉毒素变化趋势

目的探寻不同贮藏条件下花生中黄曲霉毒素含量的变化趋势。方法以远杂9102和豫花15品种的花生和花生仁为研究对象,采用免疫亲和层析净化高效液相色谱法测定其在不同储藏的条件下黄曲霉毒素的含量。结果在整个贮藏过程中,远杂9102和豫花15品种的花生和含水量低于10%的花生仁的黄曲霉毒素含量为0,而含水量高于10%的花生仁的黄曲霉毒素含量随着贮藏时间的延长而升高。在接菌量相同的条件下,同一品种的花生仁黄曲霉毒素的含量随含水量增加而增高;在含水量相同的条件下,同一品种花生仁中黄曲霉毒素的含量随接菌量的增加而增高。在相同的贮藏条件下,远杂9102花生仁中黄曲霉毒素含量极显著高于豫花15。结论在贮藏过程中,花生中黄曲霉毒素的含量与花生是否带壳、含水量、初始带菌量和品种之间具有相关性。     

Selective recovery of proteolytic strains of Clostridium botulinum types A and B from model cured meat systems

A selective medium (SBM) containing a combination of antimicrobials was developed which allowed the quantitative isolation of inoculated proteolytic Clostridium botulinum types A, B and F from a simulated cured meat product (cured pork slurry) containing natural spoilage organisms. The medium effectively suppressed the growth of Cl. perfringens, Cl. butyricum, Cl. histolyticum and non-proteolytic strains of Cl. botulinum.Other proteolytic clostridia including putrefactive anaerobes and Cl. bifermentans were capable of growth in SBM but were rarely isolated from the inoculated cured pork slurry. From unheated slurries, inoculated with Cl. botulinum type A spores and stored at 27° or 35°C 356 of 384 (92·7%) colonies picked from SBM were confirmed as Cl. botulinum type A. Selectivity was greater at 27° or 35°C than 15° or 20°C but with experience presumptive Cl. botulinum colonies can be differentiated from other resistant organisms at the lower temperatures. When heated slurries were studied all 441 colonies picked from SBM were confirmed as Cl. botulinum type A.     

Influence of long-chain polyphosphate and heat treatment on Clostridium cochlearium and Clostridium sporogenes isolated from processed cheese spread

The outgrowth of Clostridium spp. spores causes spoilage in processed cheese products due to gas and off-odor formation. The present study focuses on the response of spores of Clostridium sporogenes and Clostridium cochlearium at 25 degrees C to polyphosphate, both alone and in combination with heat treatment. The two strains used were isolated from spoiled cheese spread. The addition of 1.5% polyphosphate but not 0.75% polyphosphate totally inhibited the growth of C. sporogenes SIK4.3; in contrast, 0.75% polyphosphate was sufficient to totally inhibit C. cochlearium CCUG 45978. The highest polyphosphate concentration tested (1.5%) was sporicidal for C. sporogenes SIK4.3 but not for C. cochlearium CCUG 45978. When 0.75% polyphosphate Bekaplus FS was combined with a holding time of 5 min at 98 degrees C, no survival or growth of C. sporogenes SIK4.3 was detected; however, the same effect was not achieved through heating alone or through application of polyphosphate alone. C. cochlearium CCUG 45978 was more heat tolerant, as shown by higher D-values. In conclusion, the results strongly suggest that polyphosphate Bekaplus FS has the potential to restrict the growth of C. sporogenes and C. cochlearium in cheese spread stored at ambient storage temperature. Experiments with cheese are needed in order to verify this effect.     

Evaluation of the effect of acetylsalicylic acid on Clostridium botulinum growth and toxin production

The Republic of Georgia (ROG) has the highest incidence of botulism among all countries in the world, with most cases attributed to home-preserved vegetables. Based on epidemiologic data, the occurrence of botulism in ROG is lower in areas where aspirin (active ingredient, acetylsalicylic acid [ASA]) is added to home-canned vegetables. The objective of this study was to evaluate, with a broth medium, the antibotulinal activity of ASA to determine the possible role of ASA in preventing botulinum toxin production in home-canned vegetables. Trypticase-peptone-glucose-yeast (TPGY) broth (pH 7.0) with 0, 0.3, and 0.6 mg of ASA per ml was inoculated with a 10-strain mixture of proteolytic Clostridium botulinum type A and B spores at ca. 10(3) spores per ml. The inoculated broths were incubated at 31 degrees C under anaerobic conditions, and C. botulinum growth and botulinum toxin production were determined for up to 36 h. Results showed ASA in broth delayed (time to initial detectable toxin produced and amount of toxin produced), but did not prevent, both growth and toxin production by C. botulinum. These results would not provide a definitive explanation for differences in toxin production in canned vegetables prepared with and without aspirin.     

Antimicrobial activity of foodborne Paenibacillus and Bacillus spp. against Clostridium botulinum

The saprophytic Paenibacillus and Bacillus spp. found in cooked chilled foods may have an effect on the growth of Clostridium botulinum, a major microbiological hazard, especially for pasteurized vacuum-packaged products. Culture supernatants of 200 strains of Paenibacillus and Bacillus strains isolated from commercial cooked chilled foods containing vegetables were screened for activity against C. botulinum type A, proteolytic type B, and type E strains in a well diffusion assay. Nineteen strains were positive against C. botulinum. Among those, seven Paenibacillus polymyxa strains showed the highest antibotulinal activity and the largest antimicrobial spectrum against C. botulinum strains. The antibotulinal activity was evaluated throughout the growth of a representative strain of the positive P. polymyxa strains. The antimicrobial activity was detected in the culture supernatant from late-log/early stationary phase of the bacteria, which occurred after 7 to 10 days of incubation at 10 degrees C and after 2 to 3 days at 20 degrees C in nutrient broth and in vegetable purées under aerobic or anaerobic conditions. In co-cultures with the positive strain of P. polymyxa in nutrient broth and vegetable purées, a C. botulinum type E strain was inhibited whenever P. polymyxa reached stationary phase and produced its antimicrobial activity before C. botulinum began its exponential growth phase. The antimicrobial activity of P. polymyxa against C. botulinum was attributed to the production of antimicrobial peptides resistant to high temperature and acidity. Other gram-positive and -negative bacteria (Escherichia coli, Streptococcus mutans, Leuconostoc mesenteroides, and Bacillus subtilis) were also sensitive to these antimicrobial peptides.     

不同包装材料密闭贮藏对花生品质影响

为了获得适合贮藏花生包装材料,探讨包装材料对花生耐贮性的影响,该文研究了4种不同的包装材料密闭贮藏花生对花生色泽、品质、发芽率、虫害和黄曲霉毒素防控效果的影响。结果表明：不同包装材料贮藏花生效果差异非常显著。铝箔袋包装花生种皮颜色在贮藏期间没有发生变化,尼龙/聚乙烯(PA/PE)薄膜袋包装花生的种皮颜色变化速度比PE膜缓慢,而PE膜比编织袋包装的变化缓慢;铝箔袋、PA/PE复合膜密闭包装贮藏18个月后,花生米品质依然保持了较好;铝箔袋密闭包装能长期保持花生发芽率;4种密闭包装方式抑制虫害繁殖作用差异显著,高通透性的编织袋包装的花生害虫侵蚀严重,而铝箔袋和PA/PE袋包装可以有效地抑制虫害的发生;铝箔袋、PA/PE复合膜包装花生米原料贮藏18个月均没有黄曲霉毒素产生,但是PA/PE复合膜包装的部分花生有明显霉菌滋生。     

花生种子LOX3活力测定及其对储藏特性的影响

为测定花生中脂肪氧化酶（Lipoxygenases,LOXs;EC 1.13.11.12）活性,本研究在已有作物脂肪氧化酶LOX3β-胡萝卜素耦合比色法基础上进行优化,建立了花生LOX3活力定量测定方法,确定最适取样量8mg,最适测定时间为反应开始后10～70s。利用该方法研究214份花生种质资源的LOX3活性分布,表明材料间酶活差异极显著（P〈0.01）。对其中LOX3活力高低差异明显的24份材料进行扩繁,并在自然条件下进行18个月仓储试验,结果表明,花生LOX3活力与其种子活力呈极显著正相关（相关系数为0.6984,P〈0.01）,高LOX3活力花生种子储藏寿命也较长。该结果为进一步探讨脂肪氧化酶与花生种子储藏特性的相关性提供了依据。     

培养条件对酪酸梭菌增殖生长的影响

主要探讨了培养基组分、培养时间、接种量、接种温度等条件下酪酸梭菌的生长及其芽孢的成熟转化状况,以期在提高菌体浓度及芽孢率的同时降低生产成本。实验结果表明,通过优化外源性碳氮源及比例和无机盐的使用量等培养条件得到的最佳培养基配方为:1.5%玉米粉,1%豆粉,0.4%K2HPO4,0.05%MgSO4,0.02%MnSO4,同时,以6%的接种量,在95℃热接种及初始pH为7.0的条件下,培养36h可获得较高的生物量。最终使酪酸梭菌活菌浓度提高到基础培养基的4.7倍,芽孢成熟转化率的提高率约为52%。      

Influence of Potassium Sorbate and pH on Ten Strains of Type A and B Clostridium botulinum

Growth of each of ten strains of C. botulinum was measured as the average A_630nm of five replicate peptone-yeast extract-glucose (PYEG) broth cultures at 37°C. Media ranged in pH from 7.10–5.10 with potassium sorbate concentrations of 0–0.26%. Growth Ratios [GR = treatment/control] based on time to reach A₆₃₀=0.35 were calculated. The linear regressions of log [GR] vs log [undissociated sorbic acid in mg/L] or [1/GR] vs [pH] were used to predict GR's at 250 mg/L undissociated sorbic acid (pH 5.65 and 0.26% sorbate) and at pH 5.65 (0% sorbate) respectively. Among the ten strains examined, statistically significant differences (P<0.05) in resistance to pH and sorbate among strains and for the same strain from different spore suspensions were discovered. Resistance to pH and sorbate appeared to be correlated.