碳源诱导嗜热脂肪芽孢杆菌生产淀粉分支酶的研究

利用嗜热脂肪芽孢杆菌（Bacillus stearothermophilus）生产淀粉分支酶,研究不同碳源种类及浓度对嗜热脂肪芽孢杆菌产酶性能的影响。结果表明,以0.20%麦芽糊精为碳源时,淀粉分支酶的酶活最高,达20.02 U/mL,是其他碳源的2～10倍。采用最优培养基在小试试验的基础上进行了扩大培养中试试验,淀粉分支酶酶活稳定,为19.87 U/mL,是小试试验的1.1倍。     

Influence of recovery conditions on apparent heat resistance of Bacillus stearothermophilus spores

The effects of post-treatment environmental factors on the heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Nutrient Agar (NA), Antibiotic Assay Medium (AAM), Dextrose Tryptone Agar (DTA) and Tryptic Soy Agar (TSA) with Ca^{2+ added to a final concentration of 100 p.p.m. were used as recovery media. No significant differences were seen between D-values obtained except in the case of strain 12980 when comparing TSA with the other media and for strain 7953 comparing AAM and DTA. The optimum incubation temperature was slightly lower for heated than for unheated spores of} each strain, although, in general, 50 °C was adequate. Higher D-values were obtained at 50–55 °C. The effects of the pH of the medium in the range 5.0–7.0 and the addition of starch and phosphate on heat resistance have also been investigated. Maximum colony counts of heated spores were obtained at pH 7.0 and decreased as pH fell. D-values were significantly lower at pH ≤ 5.5. Increasing the concentration of phosphate in the recovery medium from 0 to 0.2% resulted in a progressive decrease in spore recovery and D-values. The addition of starch improved recoverability. The z-values obtained for the four strains studied under the different recovery conditions were similar with a mean value of 7.58 °C ± 0.28.     

Apparent thermal resistance of Bacillus stearothermophilus spores recovered under anaerobic conditions

 The effect of recovering Bacillus stearothermophilus spores under anaerobic conditions on their apparent thermal resistance was studied. Spores were suspended in bidistilled water as a reference medium, heated at 115, 117, 119, 121, 123 and 125°C and recovered under aerobic and anaerobic conditions. D values (decimal reduction time) obtained following recovery under anaerobic conditions were lower than those obtained under aerobic conditions. Reductions of between 31 and 48% were found for all the temperatures studied. When spores were suspended in mushroom extract and recovered under anaerobic conditions the apparent heat resistance was much lower than that obtained under aerobic conditions (D _121°C was 4.3 min and 1.7 min, under aerobic and anaerobic conditions, respectively). Heating the spores in mushroom extract and recovering the spores under anaerobic conditions produced an additive effect, decreasing the apparent heat resistance of the B. stearothermophilus spores. Received: 1 July 1997     

Bioproduction of Perillyl Alcohol and Related Monoterpenes by Isolates of Bacillus stearothermophilus

Bacillus stearothermophilus BR388 was isolated from orange peel by an enrichment culture using (+)-limonene. The thermophilic isolate exhibited growth between 45 and 68°C., with optimum growth near 55°C. BR388 could grow on limonene as a sole carbon source, but grew and degraded limonene more effectively when supplemented with small amounts of yeast extract. Perillyl alcohol was the major conversion product, with α-terpineol and perillyl aldehyde as minor products. Strains of B. stearothermophilus previously isolated from aromatic enrichments also grew on limonene, but had higher sensitivity to limonene toxicity than did BR388.     

Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis

BACKGROUND: The maltogenic amylase from Bacillus stearothermophilus (BSMA) is a valuable biocatalyst that has been used to transglycosylate natural glycosides to improve solubility. To ensure safety, BSMA was produced in Bacillus subtilis, using new shuttle vector‐based expression vectors. The transglycosylation of puerarin was also conducted with crude BSMA and analyzed. RESULTS: Two expression systems, each containing one of the promoters from the genes encoding Bacillus licheniformis maltogenic amylase (BLMA) and an α‐amylase from B. subtilis NA64 (amyR2), were constructed. The amyR2 promoter system was chosen as the best system; it yielded 107 mg of pure BSMA from a 2 L culture. In the transglycosylation reactions of puerarin using crude BSMA, relative amounts for maltosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 3)‐puerarin, and puerarin were determined as 26:18:7:49. A two‐step purification process, including gel permeation chromatography, yielded 1.7 g of the transfer products from 3 g of puerarin. CONCLUSION: The crude BSMA produced from a host generally recognized as safe (B. subtilis) can be used to transglycosylate various functional compounds. The expression system developed in this study will be helpful for the production of other food‐grade enzymes by B. subtilis. Copyright © 2010 Society of Chemical Industry     

Application of High Pressure for Spore Inactivation and Protein Denaturation

Our objective was to develop a method of sterilizing Bucillur stearothermophilus spores with minimal heating. Inactivation was achieved by spore destruction through six cycles of oscillatory pressurization at 70°C and 600 MPa, but inactivation was not complete within 60-min with continuous pressurization. Four pressurization cycles at 600 MPa and 5 min/cycle decreased the spore count from 10⁶ to 10²/mL, and six cycles decreased the count from 10⁶ to <10⁰. Spore inactivation was dependent on the adiabatic expansion velocity. On the other hand, protein denaturation by high pressures was due to phase changes of water under high pressure, and was not affected by adiabatic expansion velocity.     

Cloning and Expression of a Limonene Degradation Pathway from Bacillus stearothermophilus in Escherichia coli

The pathway encoding limonene catabolism was cloned as a 9.6kb chromosomal fragment from Bacillus stearothermophilus strain BR388 into Escherichia coli. It conferred growth on limonene as a sole carbon source and bioproduction of α-terpineol, perillyl alcohol, and perillyl aldehyde. Incubation of the recombinant with perillyl alcohol resulted in formation of perillyl aldehyde and perillic acid. Catabolic pathways apparently affected the microbial toxicity of limonene.     

耐高温β-半乳糖苷酶的分离纯化与酶学性质分析

为从嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)XG24 发酵液中纯化到β- 半乳糖苷酶,并对酶学性质进行研究,利用硫酸铵分级盐析、DEAE-Sepharose Fast Flow 阴离子交换层析和Sephadex G-75 分子筛凝胶过滤层析等方法进行分离纯化。结果表明：经过系列步骤纯化后,酶纯度提高了54.5 倍,回收率20.4%,酶比活力达32.7U/mg。以邻- 硝基酚-D- 半乳糖吡喃糖苷(ONPG)为底物,研究β- 半乳糖苷酶的酶学性质。最适pH6.5,最适作用温度65℃。此菌株产β- 半乳糖苷酶在70℃以下和pH4.0～8.0 范围内具有较好的稳定性;Mg2+、Mn2+、Fe2+和Co2+ 对此酶有明显激活作用,而Cu2+、Ag+、Hg2+ 几乎完全抑制酶活性。以ONPG 为底物酶的Km 值为4.32mmol/L。SDS-PAGE 和凝胶过滤层析测得酶蛋白为单肽链蛋白,表观分子质量64kD。因此,嗜热脂肪芽孢杆菌XG24 β- 半乳糖苷酶在乳制品工业中具有潜在的应用价值。     

肉制品主要组分对嗜热脂肪芽孢杆菌耐热性影响研究进展

嗜热脂肪芽孢杆菌是一种兼性厌氧菌,其芽孢是耐热性最强的芽孢之一,通常作为验证湿热灭菌程序的生物指示剂,同时也是造成肉制品腐败变质的主要微生物之一。本文主要从嗜热脂肪芽孢杆菌的特性、肉制品主要组分对嗜热脂肪芽孢杆菌耐热性的影响和几种“冷杀菌”方式与热杀菌的比较来介绍国内外近年来关于嗜热脂肪芽孢杆菌耐热性的研究进展。通过对这些研究成果的梳理,希望获得保障食品安全基础上的,具有风味保持、营养价值保存优点的货架期延长技术,为食品加工过程节能降耗提供新的思路。     

Inactivation of Vegetative Bacteria by Rapid Decompression Treatment

ABSTRACT: We investigated the inactivation and injury effects of hydrostatic pressure treatment combined with a slow decompression (SD treatment) and a rapid decompression (RD treatment) on several vegetative bacterial strains. Single decompression time for the SD and RD treatments was longer than 30 s and about 1 ms, respectively. The RD treatment gave significantly ( P < 0.05) smaller D and z values than the SD treatment, showing that the RD treatment was more effective than the SD treatment in inactivating vegetative bacteria and in lowering the treatment pressure. It was suggested that a rapid decompression procedure could enhance the degree of pressure-mediated injury, which caused the higher bactericidal effect of the RD treatment.     

Inhibition of Bacillus stearothermophilus Spores in a Liquid Medium by Free Fatty Acids with and without Heat: Possible Mechanisms for the Microbiological Stability of Canned Fat-Duck Liver

ABSTRACT: :
The mechanisms involved in the low-temperature sterilization of fat-duck liver (foie gras) are unclear. We have studied the antimicrobial action of its free fatty acids (FFA) on Bacillus stearothermophilus spores in liquid medium. The fat-liver FFA were identified and assayed, and their action on growth and heat resistance investigated. The major FFA were found to be palmitic, palmitoleic, stearic, linoleic, and oleic acids, the latter (C18:1) being the most abundant. The amount of these FFA in foie gras is much greater than their minimal inhibitory concentrations. They all increased thermal inactivation of B. stearothermophilus spores in our model system and so could contribute to the microbial stability of tinned foie gras.     

低温等离子体及防腐剂对解淀粉芽孢杆菌芽孢灭活效果的影响

为了研究低温等离子体及防腐剂对解淀粉芽孢杆菌（Bacillus amyloliquefaciens）芽孢杀死率的影响,分别采用不同质量浓度的单一和复合防腐剂加入到含有菌悬液的培养基中,采用不同功率与时间低温等离子体对菌体进行处理,考察不同质量浓度防腐剂及不同功率与时间低温等离子体对解淀粉芽孢杆菌芽孢致死率的影响。结果表明,单一防腐剂抑制效果由大到小顺序为脱氢乙酸钠＞乳酸链球菌素＞山梨酸钾,脱氢乙酸钠在质量浓度为4 mg/mL时,芽孢抑制率可达97%;脱氢乙酸钠∶乳酸链球菌素（1∶1）、脱氢乙酸钠∶山梨酸钾∶乳酸链球菌素（1∶2∶1）复合防腐剂抑制效果较好,质量浓度为0.5 mg/mL时芽孢抑制率可达90%以上;低温等离子体在相同功率下,时间越短,抑菌效果越好,最佳抑菌效果条件为300 W、50 s。     

Surfactin对蜡样芽孢杆菌芽孢灭活作用途径研究

目的研究Surfactin对蜡样芽孢杆菌芽孢灭活机制。方法将不同浓度Surfactin和芽孢相互作用后,利用紫外分光光度计检测芽孢内大分子组分(如蛋白质、核酸等)的泄漏情况。结果研究发现300和450μg/mL的Surfactin处理后的菌悬液中可明显检测到核酸和蛋白质的泄露。随着时间的延长,加入Surfactin后可使其DNA紫外吸收光谱发生变化,导致DNA最大吸收峰发生红移现象,最初的最大吸收峰253.5 nm移至了257 nm,产生了明显的增色效应。流式细胞的结果说明在最低抑菌浓度(minimal inhibitory concentration,MIC)的情况下,芽孢的膜结构没有明显破损,核内遗传物质没有明显泄露,随着时间延长,泄漏明显;在2～3 MIC浓度的情况下,芽孢的膜结构有明显破损,核内遗传物质有明显泄露。结论 Surfactin通过破坏芽孢壁的完整性、与DNA结合对蜡样芽孢杆菌芽孢发挥灭活效应。     

The impact of high pressure and temperature on bacterial spores: inactivation mechanisms of Bacillus subtilis above 500 MPa

Abstract: High‐pressure thermal sterilization (HPTS) is an emerging technology to produce shelf stable low acid foods. Pressures below 300 MPa can induce spore germination by triggering germination receptors. Pressures above 500 MPa could directly induce a Ca⁺²‐dipicolinic acid (DPA) release, which triggers the cortex‐lytic enzymes (CLEs). It has been argued that the activated CLEs could be inactivated under HPTS conditions. To test this claim, a wild‐type strain and 2 strains of Bacillus subtilis spores lacking germinant receptors and one of 2 CLEs were treated simultaneously from 550 to 700 MPa and 37 to 80 C (slow compression) and at 60 to 80 C up to 1 GPa (fast compression). Besides, an additional heat treatment to determine the amount of germinated cells, we added TbCl₃ to detect the amount of DPA released from the spore core via fluorescent measurement. After pressure treatment for 120 min at 550 MPa and 37 °C, no inactivation was observed for the wild‐type strain. The amount of released DPA correlated to the amount of germinated spores, but always higher compared to the belonging cell count after pressure treatment. The release of DPA and the increase of heat‐sensitive spores confirm that the inactivation mechanism during HPTS passes through the physiological states: (1) dormancy, (2) activation, and (3) inactivation. As the intensity of treatment increased, inactivation of all spore strains also strongly increased (up to ?5.7 log₁₀), and we found only a slight increase in the inactivation of one of the CLE (sleB). Furthermore, above a certain threshold pressure, temperature became the dominant influence on germination rate. Practical Application: The continuous increase of high‐pressure (HP) research over the last several decades has already generated an impressive number of commercially available HP pasteurized products. Furthermore, research helped to provoke the certification of a pressure‐assisted thermal sterilization process by the U.S. FDA in February 2009. However, this promising sterilization technology has not yet been applied in industrial settings. An improved understanding of spore inactivation mechanisms and the ability to calculate desired inactivation levels will help to make this technology available for pilot studies and commercialization at an industrial scale. Moreover, if the synergy between pressure and elevated temperature on the inactivation rate could be identified, clarification of the underlying inactivation mechanism during HP thermal sterilization could help to further optimize the process of this emerging technology.     

真空减压加速皮蛋蛋清凝胶形成的机理

真空减压法可显著缩短皮蛋加工周期,但此技术下皮蛋蛋清凝胶形成的机理还未知。因此,本文研究真空减压法下腌制皮蛋蛋清形成凝胶过程中,蛋清水分迁移、蛋白质构象变化、微观结构及分子间作用力变化等,以探究其凝胶形成机理。结果表明:在皮蛋腌制过程中,蛋清蛋白的疏水性先增加后降低,适宜的疏水相互作用有利于维持蛋白质凝胶结构的稳定;蛋清蛋白的浊度显著增大而溶解性显著降低,Zeta电位增大了1.13倍,静电斥力的增强有利于有序线性蛋白质聚集体的形成,形成了透明的凝胶;α-螺旋结构的相对含量减少至13.00%,而β-折叠和β-转角分别增加至54.00%和20.40%,蛋白质的α-螺旋结构转化为β-结构来参与蛋白质分子之间的聚集;维持蛋白质凝胶的分子间作用力从强到弱依次是是离子键、二硫键、疏水相互作用和相对较少的氢键;通过以上的相互作用,皮蛋蛋清最终形成了规则的包埋着大量结合水的三维纤维网络凝胶结构。     

Synergistic effects of high pressure processing and slightly acidic electrolysed water on the inactivation of Bacillus cereus spores

Bacillus spores are concerns for their resistance to heat, high pressure processing (HPP), and disinfectants. We examined the effects of HPP and slightly acidic electrolysed water (SAEW) on inactivation of B. cereus spores. Spores' suspensions were prepared with 2‐(N‐morpholino) ethanesulfonic acid (MES) buffer or SAEW with available chlorine content (ACC) of 24, 35, 44 or 55 mg L^?1, and then subjected to HPP. The individual effects of HPP or SAEW on spores were negligible (<1.0 log CFU mL^?1). With factorial design and anova analysis, HPP + SAEW treatment was shown to have significantly positive effects on spores’ inactivation. The optimal conditions were 300 MPa HPP + SAEW with 44 mg L^?1 ACC or 200 MPa HPP + SAEW with 44 mg L^?1 ACC + 500 MPa HPP, producing reductions of 3.27 and 3.99 log CFU mL^?1, respectively. HPP + SAEW have potentials to serve as two effective hurdle techniques for inactivating B. cereus spores.     

Comparative effect of different test methodologies on Bacillus coagulans spores inactivation kinetics in tomato pulp under isothermal conditions

Heat resistant micro‐organisms are an ongoing challenge to the food industry. Various factors may influence the heat resistance of micro‐organisms including type and strain; the environmental influences during cell and spore formations and during heat exposure; and the equipment and test tools used to perform the experimental process. In an attempt to analyse the influence of different test tools used on the heat inactivation processes, this study aimed to define the isothermal inactivation kinetics of Bacillus coagulans spores in tomato pulp at different temperatures and compare the inactivation of this bacterium when thermal death time (TDT) and capillary tube methods were used. Temperature ranges from 95 °C to 120 °C were studied, and inactivation kinetic parameters were estimated through the application of primary models. TDT inactivation curves consisted of shoulder and linear decline, while capillary method inactivation curves consisted of shoulder, linear decline and long tail. A secondary model was used to describe the influence of the temperature on spore inactivation parameters. The results showed test methods are at least as important in determining thermal processes as the micro‐organisms and media used.