复合抗冻剂的研制及对鮰鱼肌原纤维蛋白的抗冻效果

以鲫鱼鱼鳞为原料,用胰蛋白酶酶解法获得抗冻粗肽。以抗冻粗肽、海藻糖和褐藻寡糖为因素,运用{3,3}单纯形格子法进行配方设计,以冷冻24 h后嗜热链球菌的存活率为抗冻活性筛选指标,获得复合抗冻剂理论模型配方。选用复合抗冻剂最优组（K1、K2）和理论模型配方组（K3）进行冷冻鮰鱼肌原纤维蛋白试验,验证复合抗冻剂的抗冻效果。结果表明：抗冻粗肽组嗜热链球菌的存活率达57.11%。与大豆卵磷脂互作的红外光谱显示：抗冻粗肽可能与磷脂亲水头部和水之间发生相互作用,有利于稳定膜结构。K1和K2嗜热链球菌的存活率在80%以上,K3嗜热链球菌的存活率高达92%。添加K1、K2和K3复合抗冻剂后蛋白质表面疏水性显著降低（P<0.05）,蛋白质溶解度显著升高（P<0.05）。K1和K2的α-螺旋和β-折叠含量显著下降（P<0.05）,K2和K3的r值显著降低（P<0.05）。结论：理论模型K3是具有高抗冻活性,可抑制蛋白质聚集和维持蛋白质结构稳定的一种良好的水产品复合抗冻剂。本研究为复合抗冻剂的开发提供新思路,为后续开发鮰鱼预制菜冷冻产品提供了理论参考。     

抗冻多肽对嗜热链球菌冷冻干燥的保护作用

目的:研究抗冻多肽对嗜热链球菌冷冻干燥的保护作用并探讨其机理。方法:应用OD快速检测法检测冷冻干燥后的嗜热链球菌存活率,将不同浓度的抗冻多肽溶液添加到嗜热链球菌菌泥中,冷冻干燥后检测其存活率。结果:抗冻多肽能够降低嗜热链球菌冷冻干燥损伤,通过比较冻干菌粉中乳酸菌存活率得到抗冻多肽最佳添加质量浓度为1 mg/mL,最适pH 8.0,此时添加抗冻多肽的样品组存活率显著高于空白组和阳性对照组。在扫描电镜下观察嗜热链球菌冻干菌粉,添加抗冻多肽的菌体细胞饱满、完整,空白组则出现皱缩和塌陷,表明抗冻多肽对菌体细胞有保护作用。     

天然抗冻多肽的性质与对乳酸菌的低温胁迫保护作用

目的:分析猪皮明胶抗冻多肽的相关性质,研究其对乳酸菌的低温胁迫保护活性。方法:应用ANS荧光探针法测定抗冻多肽表面疏水性;应用差示扫描量热法测定抗冻多肽热滞活性;应用偏光显微镜观察低温循环过程中抗冻多肽对冰晶的重结晶抑制活性;平板菌落计数法检测低温冷冻后乳酸菌的存活率。结果:在最优条件下获得的抗冻多肽其水解度为7.2%,抗冻多肽中80%的组分分子质量分布在150～2 000 u。表面疏水性值363,等电点为3.93,热滞活性为0.58℃,有显著的重结晶抑制活性。乳酸菌低温冷冻结果表明,1%添加量的抗冻多肽,可显著提高乳酸菌存活率。     

基于分子对接技术研究鱼源抗冻多肽与鱼肌球蛋白的相互作用

冻藏期间蛋白质冷冻变性是引起解冻后鱼糜凝胶化能力下降的主要原因之一,而肌球蛋白重链（Myosin Heavy Chain,MHC）是鱼糜凝胶形成的主要贡献者。本研究使用不同生物酶酶解制备抗冻多肽,通过计算机模拟酶解技术筛选抗冻多肽。利用蛋白质同源建模构建海鲈鱼肌球蛋白空间结构,通过分子对接和分子动力学模拟解析抗冻多肽与海鲈鱼肌球蛋白重链的作用位点及可能的作用机制。结果表明,胰蛋白酶酶解物具有高抗冻活性,对菌体低温胁迫保护达到80.35%±4.39%,并具有良好的热滞活性及抑制重结晶作用。模拟胰蛋白酶酶解获得的肽段GPR与GPAGGK可以与肌球蛋白重链通过分子间作用力结合,其中GPAGGK的结合更为稳定。这种相互作用可以阻碍肌球蛋白在温度变化中的结构改变,其机理可能是抗冻多肽结合在肌球蛋白重链的结构空腔上,影响了结合水解离后引起的疏水键及二硫键等化学键的形成,阻碍蛋白侧链聚集、结构改变和冰晶的位移等,这有利于鱼糜及鱼糜制品在冷冻贮藏中的品质保持。本研究结果为抗冻多肽应用于鱼糜及其制品在冷冻贮藏过程中品质保持的应用提供科学依据。     

嗜热链球菌对生物活性肽QEPV吸收和利用的研究

以嗜热链球菌和生物活性肽Gln-Glu-Pro-Val(QEPV)为研究对象,探索了乳酸菌对生物活性肽的吸收和利用。首先,用添加荧光标记QEPV的M17培养基培养嗜热链球菌,采用流式细胞术检测QEPV能否被嗜热链球菌吸收。其次,测定不同剂量生物活性肽QEPV对嗜热链球菌活菌数、发酵液p H值、菌体浓度的影响,以探索嗜热链球菌对生物活性肽的利用情况。流式细胞术检测结果表明,FAM-QEPV与FITC-QEPV能够穿过嗜热链球菌细胞膜,推测QEPV能够被嗜热链球菌吸收、利用。并且0.5 mg/m L的QEPV能有效促进嗜热链球菌的生长繁殖,提高嗜热链球菌的发酵能力。     

食品源抗冻多肽的制备及冰晶抑制作用研究

目的:因抗冻蛋白具有控制冰晶生长,减少细胞损伤及保持产品原有组织结构、质地和品质的特点和突出意义而成为研究的热点。方法:以食品源的食用明胶为原材料,通过控制木瓜蛋白酶的切割条件,将活性多肽切割为具有特定的肽链长度和结构组成,从而使抗冻活性得以高效实现。结果:酶切多肽抗冻活性的实现受酶/底物比、酶解时间、酶解温度等条件的影响。优化的酶解条件为:pH7.0,酶/底物配比1∶10;酶解时间30min;酶解温度37℃。通过Sephadex G-50和Sephadex C-25色谱分离以及MALDI-TOF质谱鉴定,分子质量在700～1318u的亲水多肽表现显著的抗冻活性。结论:多肽酶解液在经cold-heat-stage循环后冰晶的生长和抗冻活性表现出特定的规律。控制优化酶解条件是实现多肽抗冻活性的关键所在。抗冻多肽与冰结构分子的作用机制服从表面亲水-互补作用模式。     

白鲢鱼鳞抗冻多肽的制备及对冻融鱼糜凝胶特性的改善作用

本研究以白鲢鱼鳞为原料,通过生物酶解制备白鲢鱼鳞抗冻多肽（ScAFPs）。以嗜热链球菌冻融存活率为主要指标,以水解度为辅助指标,通过单因素实验及响应面试验优化,得到ScAFPs最佳酶解制备工艺;同时进行ScAFPs的基本性质及其对鱼糜凝胶稳定性影响的研究。结果表明,选用的酶制剂为胰蛋白酶、底物浓度5.0%、酶底比3.8%、酶解温度37 oC、酶解时间3.5 h,此条件下制备的ScAFPs对嗜热链球菌冷冻存活率为82.19%±1.03%,水解度为7.54%±0.43%。ScAFPs相对分子量主要集中于180~3000 Da之间,等电点在4.2左右,且具有极强的亲水性和较好的热稳定性,能有效降低体系冰晶含量。ScAFPs对冻融鱼糜的凝胶特性的影响结果表明,经过5次冻融循环后,ScAFPs处理组鱼糜凝胶白度、硬度、咀嚼性、凝胶强度等指标下降幅度均显著（P<0.05）低于未添加低温保护剂的鱼糜组,且当ScAFPs添加量超过2%时,ScAFPs对鱼糜的冻融保护效果优于商业抗冻剂（4%蔗糖与4%山梨醇混合物）。本研究为利用白鲢鱼鳞加工副产物开发新型低温保护剂并探索在冷冻鱼糜及凝胶制品的应用奠定理论基础。     

冷冻面团中酵母的抗冻性及抗冻酵母的研究进展

随着烘焙工业中冷冻面团技术的引进和迅速发展．酵母的抗冻性作为冷冻面团的一个主要影响因素,也应引起关注。文章综述了对冷冻面团中酵母抗冻性的研究进展,从物理和生物学的角度分析了温度变化对酵母抗冻性的影响,冷冻过程中介质中和细胞内部冰晶的形成是造成细胞损伤的主要物理因素。探讨了抗冻酵母的选育和培育方法,如调整培养条件选育抗冻酵母、生物技术培育抗冻酵母等。总之,要想得到真正的抗冻酵母,就要从酵母冷冻过程中的变化和抗冻机理研究,为抗冻酵母的工业生产提供理论指导。     

谷朊抗冻多肽对面团冷冻保护作用研究

采用旋转流动仪、SDS-PAGE凝胶电泳和原位拉曼光谱等手段探究谷朊抗冻多肽对面团流变特性、蛋白分子质量分布和分子结构的影响规律。结果表明，经过冷冻(冻融和冻藏)处理后，与对照相比，加入谷朊抗冻多肽的面团黏弹性有所下降，谷朊抗冻多肽可以抑制冷冻过程中GMP的解聚。谷朊抗冻多肽的添加有效维持了面筋蛋白的二级结构和分子排列的有序性，与对照相比，在经过反复冻融和恒温冻藏处理后，α-螺旋结构的质量分数分别提高了3.1%、2.5%,二硫键g-g-g构型质量分数分别提高了8.8%、9.8%,同时使得氨基酸侧链中I₈₅₀/I₈₃₀值下降、I₇₆₀值提高。当谷朊抗冻多肽的质量分数为0.5%时最有效地抑制了冷冻过程中冰晶生长对面筋蛋白网络结构的破坏，谷朊抗冻多肽保护了面筋网络结构的完整性，维持了面筋蛋白在面团中的骨架作用。     

青钱柳多糖抑菌活性及及作用机理

为研究青钱柳多糖的抑菌活性和作用机制，用不同质量浓度的青钱柳多糖处理细菌，通过最小抑菌浓度（minimum inhibitory concentration, MIC）、抑菌生长曲线分析青钱柳多糖对细菌的抑菌效果，并从抑制效果最佳的受试菌菌体电导率、细胞蛋白和核酸含量、还原糖的释放、呼吸链脱氢酶活性、脂质过氧化程度、细胞内ATP含量、细胞外碱性磷酸酶活力的角度来揭示其抑菌机理。结果表明，青钱柳多糖对金黄色葡萄球菌（Staphylococcus aureus）、大肠杆菌（Escherichia coil）、单增李斯特氏菌（Listeria monocytogenes）和鼠伤寒沙门氏菌（Salmonella typhimurium）有明显的抑制作用，MIC分别为2、4、6、6 mg/mL，对枯草芽孢杆菌（Bacillus subtilis）的抑菌性不明显；对指示菌的电导率、蛋白质和核酸渗漏、还原糖释放、呼吸链脱氢酶活性、细菌脂质过氧化程度、菌体胞内ATP和胞外碱性磷酸酶（AKP）含量均有影响。以上现象表明，青钱柳多糖的抑菌性主要是破坏菌体细胞壁和细胞膜完整性，改变其通透性，使得胞内大小分子物质外泄，抑制了细胞的呼吸代谢和引起氧化损失，进而影响细胞的生长代谢或造成细胞死亡，达到抑菌效果。     

The structures and functions of ice crystal-controlling proteins from bacteria

Many organisms have evolved into unique mechanisms which minimize freezing injury due to extracellular ice formation. Specifically, certain bacteria have produced a few proteins each with different functions. For example, the ice nucleation protein acts as a template for ice formation, which is responsible for imparting ice nucleating activity. The anti-nucleating protein inhibits the fluctuation of ice nucleus formation by a foreign particle in the water drop. Also, the antifreeze proteins depress the freezing temperature, modify or suppress ice crystal growth, inhibit ice recrystallization, and protect the cell membrane from cold-induced damage. In this article, a review on the current knowledge of the structure and the function of these three types of proteins, which are capable of interacting with ice itself or its nuclei from bacteria.     

Resistance to freezing and frozen storage of Streptococcus thermophilus is related to membrane fatty acid composition.

The resistance to freezing and frozen storage of Streptococcus thermophilus was related to the fatty acid composition of the cell membrane. The effects of four experimental factors were investigated on the fatty acid concentrations and on the recovery of acidification activity of S. thermophilus stored at -20 degrees C by using a complete experimental design: incorporating oleic acid in the culture medium, fermentation pH, addition of glycerol as cryoprotective agent and duration of storage. The acidification activity decreased during the freezing and the frozen storage of S. thermophilus. The storage time slightly enhanced the unsaturated fatty acid concentrations. The addition of glycerol did not modify the fatty acid composition but increased the resistance to frozen storage. The addition of oleic acid and the decrease of the fermentation pH enhanced the ratio unsaturated:saturated fatty acids and improved the recovery of the acidification activity. These results indicate that the resistance to frozen storage was closely related to the membrane fatty acid composition. We interpreted this as an adaptation of S. thermophilus to the addition of oleic acid and the unfavorable growth conditions that corresponded to a low fermentation pH.     

丁香酚复合保鲜剂对腐败希瓦氏菌的抗菌作用机制

目的：探讨丁香酚复合保鲜剂对水产品优势腐败菌的抗菌性能和作用机制。方法：以腐败希瓦氏菌为抗菌对象,测定丁香酚、麝香草酚、乳酸、溶菌酶（食品级,18 U/mg）、溶菌酶（生物级,20 000 U/mg）及丁香酚/麝香草酚、丁香酚/乳酸、丁香酚/溶菌酶（食品级）和丁香酚/溶菌酶（生物级）的抗菌性能,采用扫描电子显微镜和透射电子显微镜观察丁香酚及其复合保鲜剂处理前后菌体的微观形态变化,同时测定菌体细胞膜的完整性、通透性、损伤程度及菌体内Na＋、K＋-腺苷三磷酸酶（adenosine triphosphatase,ATPase）和碱性磷酸酶（alkaline phosphatase,AKPase）活力。结果：丁香酚对腐败希瓦氏菌有较好的抗菌性能,可使菌体形态发生扭曲、皱缩、塌陷,细胞壁膜溶解。丁香酚对菌体细胞膜结构造成了一定程度的损伤,导致膜完整性变差、膜通透性增大,Na＋、K＋-ATPase和AKPase活性降低;丁香酚与麝香草酚、乳酸、食品级溶菌酶和生物级溶菌酶复配后,呈现出协同抗菌作用,使菌体结构损伤更加严重,细胞膜完整性更差,通透性更大,Na＋、K＋-ATPase和AKPase活性显著降低（P＜0.05）。结论：研究可为水产品加工贮藏过程中新型保鲜剂研发提供参考。     

海藻糖与酿酒酵母乙醇耐受性相关性的研究进展

为了得到具有高富锌能力的酵母菌,该研究以面包来源的酵母菌DLY28为出发菌株,重复驯化后筛选获得一株优良耐锌酵母,并通过单因素试验及响应面试验对其富锌培养基进行优化。结果表明,通过驯化筛选得到一株优良耐锌酵母S7,其富锌的最优培养基组成为蔗糖含量82 g/L、胰蛋白胨含量26 g/L、锌含量404 mg/L。在此最优条件下,富锌酵母S7的锌吸附量为18.79 mg/g,生物量（OD600 nm值）为1.49。该研究为富锌酵母的生产以及食品有机锌的开发应用提供理论依据。     

Damage to Pork Liver Caused by Repeated Freeze-Thaw Cycling and Refrigerated Storage

The effects of freezing and thawing (F-T) and refrigerated storage (R-S) on pork liver were examined. Intact cells were isolated from the tissue and analyzed for membrane integrity and the tissue extracts were analyzed for protein content. Significant (p < 0.05) differences were noted in the number of isolated intact cells and in tissue protein content between F-T and R-S or fresh livers. Differences in cell membrane integrity were also noted between fresh and R-S livers. Repeated freezing and thawing appears to break down the liver structure differently than does refrigerated storage.     

Rapid assessment of the physiological status of Streptococcus macedonicus by flow cytometry and fluorescence probes

Flow cytometry in combination with fluorescence probes was applied to rapidly assess the physiological status of Streptococcus macedonicus ACA-DC 198, a newly described member of the lactic acid bacteria group with technologically important features (e.g. lantibiotic production). A sonication procedure was developed for disaggregating typical streptococci chains in order to optimize cell preparations for single cell analysis. Single stained live and dead populations of S. macedonicus cells were clearly resolved based on membrane potential by bis-oxonol [DiBAC(4)(3)], membrane integrity by Propidium Iodide (PI) and enzymatic activity as well as membrane integrity by Carboxyfluorescein Diacetate (cFDA). Further, estimation of both live and dead cells by a cFDA/PI two-colour flow cytometric assay showed excellent correlation with the dead cells in the samples (dead(FCM)=0.9945 dead(S)-0.806, R(2)=0.9986 and live(FCM)=-0.978 dead(S)+98.895, R(2)=0.9992). Finally, the assay was applied to study the physiology of S. macedonicus after acid stress. Interestingly, in situ assessment of the physiological status of stressed S. macedonicus cells by flow cytometry and single cell sorting revealed the coexistence of three distinct subpopulations according to their fluorescence labelling behaviour and culturability, representing intact/culturable, permeabilized/dead and potentially injured cells with the latter exhibiting both metabolic activity and membrane permeabilization as well as decreased culturability.     

Study of Pulsed Light-Induced Damage on Saccharomyces cerevisiae in Apple Juice by Flow Cytometry and Transmission Electron Microscopy

This work analyzed the pulsed light (PL) (0.0–71.6 J/cm²)-induced damage on Saccharomyces cerevisiae KE162 cells in peptone water (pH 3.5 or 5.6) and apple juice (pH 3.5) by applying flow cytometry (FCM) and transmission electronic microscopy. Cells were labeled with fluorescein diacetate (FDA) for detecting membrane integrity and esterase activity and with propidium iodide (PI) for monitoring membrane integrity. S. cerevisiae inactivation curves reached 6–7 log reductions (peptone water systems) and 3.9 log reductions (apple juice) after 60 s (71.6 J/cm²) of PL exposure. FCM revealed the same damage pattern (although at different doses) in all media: at low doses, there was an increase in population in PI⁺?FDA⁺ quadrant, while at high doses, most of the population was located at quadrant PI⁺–FDA^?, indicating that PL provoked rupture of the cytoplasm membrane allowing PI to penetrate cells and there was progressive loss of esterase activity. Comparison of conventional culture technique with FCM revealed the occurrence of certain cell subpopulations in peptone water with pH 3.5 which were stressed and lost their ability to grow in agar but still showed metabolic activity. Transmission electron microphotographs of PL-treated cells clearly indicated that various cell structures other than plasma membranes were altered and/or destroyed in a different degree depending on exposure time and type of medium.     

南极磷虾酶解工艺优化及酶解产物对牡蛎肉的冷冻保护作用

为了开发安全高效的无磷抗冻剂,本研究以南极磷虾为原料制备酶解产物对其进行冷冻保护作用评价,为开发新型抗冻剂提供基础数据。以水解度为指标,对南极磷虾进行酶解,从4种蛋白酶中筛选得到碱性蛋白酶作为实验用酶,在单因素实验的基础上结合正交试验对酶解工艺进行了优化;并以解冻失水率、盐溶性蛋白含量、总巯基含量、Ca2+-ATPase酶活力为指标,考察了南极磷虾酶解产物对牡蛎肉的冷冻保护作用。结果表明,用碱性蛋白酶酶解南极磷虾的最佳条件为温度为55℃,酶解pH为8.5,加酶量2.6%,酶解时间为5 h;在此工艺参数下南极磷虾酶解产物相对分子质量主要分布在100~5500 Da,占总酶解产物的79.69%。南极磷虾酶解产物可以抑制牡蛎冻藏后失水,延缓盐溶性蛋白、Ca2+-ATPase酶活力、总巯基含量下降,其作用效果优于含磷抗冻剂。因此,南极磷虾酶解产物具有冷冻保护活性,可进一步对其进行分离鉴定研究。     

酸奶制品发生后酸化主要发酵剂菌确定及性质研究

将培养物中保加利亚乳杆菌与嗜热链球菌调节为相同 pH值 ( 4 .5 0 )、起始相同菌数7.4× 1 0 7个 /mL后 ,分别贮存 ( 2 5℃ ) ,贮存第 1 1d ,嗜热链球菌的 pH值为 4.5 7,保加利亚乳杆菌的为 3.85。将保加利亚乳杆菌确定为导致后酸化发生的主要发酵剂菌 ,是其细胞壁或细胞膜的性质保护了乳糖酶活性     

基于组织蛋白酶催化的不冻液冻结中华管鞭虾中肌原纤维蛋白氧化分析

对比分析利用不冻液(-18℃和-30℃)冻结后冻藏与直接置于冰箱(-18℃和-30℃)冻结后冻藏的中华管鞭虾肌原纤维蛋白氧化指标变化以及组织蛋白酶B、H、L活性变化,通过肌原纤维微结构观察,分析酶活性对肌原纤维蛋白水解产生的作用,并探讨其与肌组织结构的相关性。结果表明：采用-18℃和-30℃不冻液冻结的中华管鞭虾的盐溶性蛋白含量(92.7 mg/g和97.8 mg/g)均明显高于-18℃和-30℃冰箱缓冻组(72.40 mg/g和77.90 mg/g);-30℃不冻液组的表面疏水性最低(400.6μg);两种冻结方式下-30℃组Ca²⁺-ATPase活性均高于-18℃组;总巯基含量变化趋势与Ca²⁺-ATPase活性变化趋势一致。冻藏前期(0～60 d),低温不冻液冻藏能较好地抑制中华管鞭虾组织蛋白酶活性,使其蛋白结构保持良好;冻藏后期(60～120 d),其对于酶活性的抑制作用减弱。综合以上结果,在一定冻藏期内,不冻液冻结比冰箱冻结能够更好减弱冻藏过程中中华管鞭虾的肌原纤维蛋白的氧化和组织蛋白酶活性,且不冻液的冻结温度越低,冻结速率越高,组...