生孢梭菌芽孢萌发条件的优化

本文研究了不同温度、时间和pH值条件下,生孢梭菌芽孢萌发所需的最佳条件.本研究将活化传代好的生孢梭菌菌液培养7 d,多次离心后,加入无菌水制成生孢梭菌芽孢悬浮液,处理温度为70℃～90℃,处理时间为5～25 min,处理pH值为4～8,用平板计数法观察计算不同条件处理后的芽孢萌发率,用响应面优化法对影响生孢梭菌芽孢萌发...     

Germination and outgrowth of spores of Bacillus cereus group members: diversity and role of germinant receptors

Bacillus cereus is a Gram-positive, facultative anaerobic, endospore-forming toxicogenic human pathogen. Endospores are highly specialized, metabolically dormant cell types that are resistant to extreme environmental conditions, including heat, dehydration and other physical stresses. B. cereus can enter a range of environments, and can in its spore form, survive harsh conditions. If these conditions become favorable, spores can germinate and grow out and reach considerable numbers in a range of environments including processed foods. Certainly the last decade, when consumer preferences have shifted to mildly processed food, new opportunities arose for spore-forming spoilage and pathogenic organisms. Only rigorous methods have been shown to be capable of destroying all spores present in food, thus a shift toward e.g., milder heat preservation strategies, may result in low but significant amounts of viable spores in food products. Hence, the need for a mild spore destruction strategy is eminent including control of spore outgrowth. Consequently, there is a large interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. Another option could be to prevent germination so that no dangerous levels can be reached. This contribution will focus on germination and outgrowth characteristics of B. cereus and other members of the B. cereus group, providing an overview of the niches these spore-formers can occupy, the signals that trigger germination, and how B. cereus copes with these wake-up calls in different environments including foods, during food processing and upon interaction with the human host.     

Inhibition of Spore Germination of Phakopsora Pachyrhizi Using Crude Extracts of Amaranthus Spinosus

The effectiveness of methanolic and n-hexane crude extracts of Amaranthus spinosus to inhibit the spore germination of Phakopsora pachyrhizi, a causal agent of soybean rust disease was studied. Both methanolic and n-hexane crude extracts inhibited spore germination at concentrations of 0.1% to 5.0%. Methanolic extract of the roots at a concentration of 2.5% inhibited 55% of spore germination, and this result was similar when higher concentration at 5% of methanolic and n-hexane extracts of the flowers was used. The presence of alkaloids, flavonoids, tannins, saponins, and terpenoids in the extract may be responsible for the inhibition. The extract was potential to be utilized as a botanical fungicide.     

不同浓度芽孢萌发剂对肉毒梭菌芽孢萌发的影响

肉毒梭菌（Clostridium botulinum）及芽孢是真空包装、冷冻食品及罐装食品中的主要杀菌对象,其抗逆性强,生长速度快,而成为食品中毒的主要原因之一。本试验采用单因素试验和响应面分析法研究芽孢萌发剂L-丙氨酸和KCl添加浓度,处理温度和处理时间对肉毒梭菌芽孢萌发条件的影响,探究影响肉毒梭菌芽孢萌最适条件,并结合常压100 ℃,20 min杀菌,研究最终肉毒梭菌的杀灭效果。结果显示,当处理温度为75.5 ℃,处理时间为25.0 min,添加L-丙氨酸浓度为15.4 mmol/L,KCl浓度为0.53%时,芽孢萌发率最大,可达89.91% ± 1.31%,经过两次100 ℃杀菌20 min,肉毒梭菌杀灭率可达98%。研究结果可为食品工业中罐头的常温杀菌提供一定的技术指导。     

钾离子通道在黄曲霉菌孢子萌发中的作用

黄曲霉毒素严重污染农产品,威胁人畜生命健康,而孢子萌发是黄曲霉菌生存及侵染过程中的决定性事件,从最上游阻断孢子萌发可能是有效控制黄曲霉污染、开发新型抗菌药物的突破口之一。研究发现一定浓度的胞外KCl能够影响黄曲霉孢子的萌发。通过使用K+特异性荧光探针PBFI-AM,发现适合孢子萌发的特定浓度K+能够激发黄曲霉孢子瞬间产生较高的钾离子流,说明K+响应可能是黄曲霉孢子萌发的早期信号。4-AP作为特异性电压门控钾离子通道阻滞剂,能够明显抑制并延迟孢子的萌发。通过生物信息学预测,发现黄曲霉菌KCNA具有钾离子通道选择器序列,具备电压门控钾离子通道的α亚基属性。进一步构建KCNA非洲爪蟾卵母细胞表达体系,用双电极电压钳系统测得KCNA外向型电流,一定程度上证明了KCNA电压门控钾离子通道蛋白质属性,也说明电压门控钾离子通道参与了黄曲霉孢子萌发。     

不同菌体肽聚糖对产气荚膜梭菌芽孢萌发的诱导作用

为了研究不同菌体肽聚糖对芽孢萌发的影响,本研究分别提取了产气荚膜梭菌(Clostridium perfringens,C. perfringens)、枯草芽孢杆菌(Bacillus subtilis,B. subtilis)及金黄色葡萄球菌(Staphylococcus aureus,S. aureus)细胞壁肽聚糖,以C. perfringens芽孢为研究对象,按照1:10、1:100、1:1000比例分别加入提取的三种菌体肽聚糖,并以芽孢浑浊度(OD_(600))、芽孢折光性、2,6-吡啶二羧酸(DPA)的释放量、热抗性损失等指标比较不同菌体肽聚糖对C.perfringens芽孢萌发的效果。结果显示,按照一定百分比分别加入三种菌体肽聚糖,1:10比例添加C. perfringens营养体肽聚糖诱导C. perfringens芽孢萌发效果最佳,在60 min后,其芽孢浑浊度(OD_(600))下降了31.71%,DPA释放率上升可达到38.29%;1:10比例添加B. subtilis营养体肽聚糖诱导C. perfringens芽孢萌发效果次之,其芽孢浑浊度(OD_(600))下降了24.92%,DPA释放率上升可达到30.84%;而不同比例S. aureus营养体肽聚糖对C. perfringens芽孢萌发均无显著影响。本研究可为进一步阐明肽聚糖诱导芽孢萌发的机制提供重要信息。     

高压静电场对青霉菌的影响

以苹果霉腐病中青霉菌为材料通过高压静电场(HVEF)进行不同时间的处理,比较处理的孢子菌悬液在不同培养温度下的萌发和产孢量,将处理后的孢子菌悬液接种于苹果,观察苹果的感病率及病斑直径。结果表明：同一温度下培养,高压静电场处理的与未处理菌悬液的孢子萌发率相差不大,但萌发时间滞后;产孢量与处理时间相关,处理30min 后,在20℃培养7d 产孢量要比处理15min 和未处理的低。与对照相比,接种处理的孢子菌悬液的苹果感病率较低,病斑直径较小。     

Effect of Pulsed Light Treatment on the Germination of Bacillus subtilis Spores

Food and Bioprocess Technology - The loss of cell culturability (spore counts), spore germination and subsequent vegetative growth of Bacillus subtilis were evaluated after pulsed light (PL)...     

Inhibition of Clostridium perfringens Spore Germination and Outgrowth by Lemon Juice and Vinegar Product in Reduced NaCl Roast Beef

Abstract: Inhibition of Clostridium perfringens spore germination and outgrowth in reduced sodium roast beef by a blend of buffered lemon juice concentrate and vinegar (MoStatin LV1) during abusive exponential cooling was evaluated. Roast beef containing salt (NaCl; 1%, 1.5%, or 2%, w/w), blend of sodium pyro‐ and poly‐phosphates (0.3%), and MoStatin LV1 (0%, 2%, or 2.5%) was inoculated with a 3‐strain C. perfringens spore cocktail to achieve final spore population of 2.5 to 3.0 log CFU/g. The inoculated products were heat treated and cooled exponentially from 54.4 to 4.4 °C within 6.5, 9, 12, 15, 18, or 21 h. Cooling of roast beef (2.0% NaCl) within 6.5 and 9 h resulted in <1.0 log CFU/g increase in C. perfringens spore germination and outgrowth, whereas reducing the salt concentration to 1.5% and 1.0% resulted in >1.0 log CFU/g increase for cooling times longer than 9 h (1.1 and 2.2 log CFU/g, respectively). Incorporation of MoStatin LV1 into the roast beef formulation minimized the C. perfringens spore germination and outgrowth to <1.0 log CFU/g, regardless of the salt concentration and the cooling time. Practical Application: Cooked, ready‐to‐eat meat products should be cooled rapidly to reduce the risk of Clostridium perfringens spore germination and outgrowth. Meat processors are reducing the sodium chloride content of the processed meats as a consequence of the dietary recommendations. Sodium chloride reduces the risk of C. perfringens spore germination and outgrowth in meat products. Antimicrobials that contribute minimally to the sodium content of the product should be incorporated into processed meats to assure food safety. Buffered lemon juice and vinegar can be incorporated into meat product formulations to reduce the risk of C. perfringens spore germination and outgrowth during abusive cooling.     

Germination of Bacillus cereus spores adhered to stainless steel

Adhered spores of Bacillus cereus represent a significant part of the surface-derived contamination in processing equipment used in the dairy industry. As germinated spores lose their resistance capacities instantaneously, efficient germination prior to a cleaning in place treatment could aid to the disinfecting effect of such a treatment. Therefore, spores of B. cereus ATCC 14579 and that of the environmental isolate B. cereus CMCC 3328 were assessed for their germination behaviour when adhered to a stainless steel surface. A mixture of l-alanine and inosine initiated germination of adhered spores efficiently, resulting in 3.2 decimal logarithms of germination. Notably, implementation of a germination-inducing step prior to a representative cleaning in place procedure reduced the number of survivors with over 3 decimal log units, while an alkali treatment alone, as part of the cleaning in place procedure, did not show any effect on B. cereus spore viability. These results show that implementation of a germination step enhances the disinfection effect of currently used cleaning in place procedures.     

Sporulation and Germination Gene Expression Analysis of Bacillus anthracis Sterne Spores in Skim Milk under Heat and Different Intervention Techniques

ABSTRACT:  To investigate how B. anthracis Sterne spores survive in milk under heat (80 °C, 10 min), pasteurization (72 °C, 15 s), microfiltration, and pasteurization and microfiltration, the expression levels of genes related to sporulation and germination were tested using real-time PCR assays. Twenty-seven sporulation- and germination-related genes were selected for the target genes. Our results demonstrated that gene expression levels were altered by heat and microfiltration whereas the pasteurization and pasteurization and microfiltration resulted in less alteration of gene expression. Heat activated and inhibited both sporulation- and germination-related genes, suggesting that bacterial spores underwent different molecular mechanism for heat treatments. Our results may provide some insight into the molecular mechanisms of spore survival in response to heat treatment and different intervention strategies used to treat fluid skim milk.     

Inactivation of Bacillus cereus spores in milk by mild pressure and heat treatments

The objective of this work was to study the germination and subsequent inactivation of Bacillus cereus spores in milk by mild hydrostatic pressure treatment. In an introductory experiment with strain LMG6910 treated at 40 degrees C for 30 min at 0, 100, 300 and 600 MPa, germination levels were 1.5 to 3 logs higher in milk than in 100 mM potassium phosphate buffer (pH 6.7). The effects of pressure and germination-inducing components present in the milk on spore germination were synergistic. More detailed experiments were conducted in milk at a range of pressures between 100 and 600 MPa at temperatures between 30 and 60 degrees C to identify treatments that allow a 6 log inactivation of B. cereus spores. The mildest treatment resulting in a 6 log germination was 30 min at 200 MPa/40 degrees C. Lower treatment pressures or temperatures resulted in considerably less germination, and higher pressures and temperatures further increased germination, but a small fraction of spores always remained ungerminated. Further, not all germinated spores were inactivated by the pressure treatment, even under the most severe conditions (600 MPa/60 degrees C). Two possible approaches to achieve a 6 log spore inactivation were identified, and validated in three additional B. cereus strains. The first is a single step treatment at 500 MPa/60 degrees C for 30 min, the second is a two-step treatment consisting of pressure treatment for 30 min at 200 MPa/45 degrees C to induce spore germination, followed by mild heat treatment at 60 degrees C for 10 min to kill the germinated spores. Reduction of the pressurization time to 15 min still allows a 5 log inactivation. These results illustrate the potential of high-pressure treatment to inactivate bacterial spores in minimally processed foods.     

MODELING OF THE GERMINATION OF SPORES FROM CLOSTRIDIUM PERFRINGENS FOOD POISONING ISOLATES

Clostridium perfringens type A isolates carrying a chromosomal gene for enterotoxin production (chromosomal c. perfringens enterotoxin [ C- cpe] strains) are frequent causative agents of food poisoning. Predictive models for their growth in meat products have been published; however, the development of germination models needs further research. In this study, the Weibull function was used to describe the germination of C- cpe food poisoning strains in phosphate and Tris–HCl buffer as affected by pH (5.8–8.5), germinant concentration (1–100 mM KCl) and germination temperature (23–60C). As indicated by estimators of model accuracy and bias, an empirical model for spore germination as a function of germination temperature only, predicted accurately the germination potential of 4 C- cpe food poisoning strains in buffer and in laboratory media in the 23–50C range.

PRACTICAL APPLICATIONS

Thermal processing models based on the inactivation of a heat-resistant spore ignore the potential germination and outgrowth of spore survivors as recommended by new food safety guidelines. Germination models for spore survivors to be used in conjunction with outgrowth models should be developed using as samples the product of interest because the bioavailability and concentration of germinants such as ions and free amino acids vary with the product and its manufacturing process. Another germination model application is in conjunction with new processing technologies such as high pressure processing (HPP). The inactivation of bacterial spores has been a major challenge to HPP process developers. Since germinated spores are sensitive to pressure, germination models would be useful to design combined strategies of food thermal treatment and HPP.     

Germination, Growth, and Toxin Production of Nonproteolytic Clostridium botulinum as Affected by Multiple Barriers

ABSTRACT The effect of combinations of pH (6.5, 5.75), NaCl (0.25,1.75%), and incubation temperatures (7,13 °C) on spore germination, outgrowth, and time to toxicity of nonproteolytic Clostridium botulinum was examined in a broth system. Spores of four toxin type E and nonproteolytic type B were inoculated (10⁴/ml) into Tryptone Peptone Glucose Yeast Extract (TPGY) broth. Cultures were monitored for three weeks, or until toxin was detected. A modified FSIS‐amplified ELISA (comparable in sensitivity to the mouse bioassay) was used to screen cultures for neurotoxin. Combinations of the most inhibitory level for each barrier reduced the degree and rate of germination, the lag and growth rate of vegetative cells, and the time to toxicity.     

Does proximity to neighbours affect germination of spores of non-proteolytic Clostridium botulinum?

It is recognised that inoculum size affects the rate and extent of bacterial spore germination. It has been proposed that this is due to spores interacting: molecules released from germinated spores trigger germination of dormant neighbours. This study investigated whether changes to the total number of spores in a system or proximity to other spores (local spore density) had a more significant effect on interaction between spores of non-proteolytic Clostridium botulinum strain Eklund 17B attached to defined areas of microscope slides. Both the number of spores attached to the slides and local spore density (number of spores per mm²) were varied by a factor of nine. Germination was observed microscopically at 15 °C for 8 h and the probability of, and time to, germination calculated from image analysis measurements. Statistical analysis revealed that the effect of total spore number on the probability of germination within 8 h was more significant than that of proximity to neighbours (local spore density); its influence on germination probability was approximately four-times greater. Total spore number had an even more significant affect on time to germination; it had a nine-fold greater influence than proximity to neighbours. The applied models provide a means to characterise, quantitatively, the effect of the total spore number on spore germination relative to the effect of proximity to neighbouring spores.     

大豆萌发过程中异黄酮变化及提升策略

异黄酮在人体内具有丰富的生理活性，被证明在抗氧化及预防乳腺癌、前列腺癌、骨质疏松症和心血管疾病等方面都有积极的作用，因而逐渐受到功能性食品开发的青睐。萌发是改善植物中营养价值的常见手段，而大量研究结果表明，胁迫环境下的萌发能进一步的提升大豆中异黄酮含量、改善其异黄酮组成。因此多种胁迫萌发的条件被用于评估大豆异黄酮含量的提升效果。本文阐述了萌发以及萌发的环境因素对大豆异黄酮含量的影响以及萌发过程中豆芽不同部位中异黄酮含量的变化差异，并重点介绍了多种提升豆芽异黄酮含量的胁迫策略。以期能够为功能性食品的开发提供帮助，并更加全面了解该领域的研究进展。     

亚损伤状态下枯草芽孢杆菌芽胞萌芽影响因素的研究

为了研究食品中营养要素对处于亚损伤状态枯草芽孢杆菌芽胞的影响,采用分批培养法,利用食品中存在的碳源、氮源、无机盐离子等分别诱导芽胞萌芽,采用正交实验研究了温度、p H、接种量、溶氧量对芽胞萌芽率的影响,分别在OD580下测定其光密度值,得到芽胞萌芽率。结果显示,麦芽糖、乳清蛋白是芽胞萌芽的最佳碳源、氮源;无机盐Mg SO4对芽胞萌芽产生显著的诱导作用。以麦芽糖（5 mg/m L）,Mg SO₄（5 mg/m L）,乳清蛋白（5 mg/m L）为基础培养基,芽胞的最佳生长条件为:接种量4%、p H7.0、溶氧量70 m L,37℃恒温振荡培养60 min,得芽胞萌芽率最大为15.27%。      

Variation of the spore population of a natural source strain of Bacillus cereus in the presence of inosine

The heat resistance of a wild strain of Bacillus cereus spores isolated from liquid egg was characterized, and the effect of the nutritional germinant inosine on the spore population was then studied, considering different factors such as germination temperature, inosine concentration, and age of spore culture. The heat resistance clearly indicates that these spores can survive mild heat treatments such as those used for cooked refrigerated food of extended durability or liquid egg, posing safety problems for these foods with temperature abuse. The germination study indicates that temperature, spore age, and the interaction between the two were the factors affecting the level of spores remaining after the germination process. No significant differences were found for the three inosine concentrations used in the study (1, 5, and 10 mM). The highest reduction in the spore concentration was reached at 30 degrees C after 120 min, although the reduction in the spore counts at germination temperatures of 4 and 8 degrees C was also considerable.     

Promoting Bacillus cereus spore germination for subsequent inactivation by mild heat treatment

Sublethal heat treatment may activate dormant spores and thereby potentiate the conversion of spores to vegetative cells. As the germinated spore is known to possess lower heat resistance than its dormant counterpart, it has been postulated that double heat treatment, i.e., spore heat activation followed by germination and then by heat inactivation, can be used to control spores in foods. Production of refrigerated processed foods of extended durability often includes more than one heat treatment of the food components. This work simulates conventional heat treatment procedures and evaluates double heat treatment as a method to improve spore control in model food matrixes of meat broth and cream sauce. Bacillus cereus NVH 1230-88 spores were supplemented in food model matrixes and heat activated at 70°C and then heat inactivated at 80 or 90°C. The samples were held at 29 to 30°C for 1 h between primary and secondary heat treatments, to allow spore germination. Nutrients naturally present in the food matrixes, e.g., amino acids and inosine, could act as germinants that induce germination. The levels of germinants could be too low to produce effective germination within 1 h. Following primary heat treatment, some samples were therefore supplemented with a combination of L-alanine and inosine, a germinant mixture known to be effective for B. cereus spores. In both matrixes, a combination of double heat treatment (heat activation, germination, and inactivation) and addition of germinants gave a reduction in spore counts equivalent to or greater than that obtained with a single heat treatment for 12 min at 90°C. Addition of germinants was essential to induce effective germination in cream sauce during 1 h at 29 to 30°C, and germinants were therefore a crucial supplement to obtain an effect of double heat treatment in this matrix. These data will be valuable when setting up temperature-time-germinant combinations for an optimized spore reduction in mild-heat-treated foods.