Antifreeze Proteins: Novel Applications and Navigation towards Their Clinical Application in Cryobanking

Antifreeze proteins (AFPs) or thermal hysteresis (TH) proteins are biomolecular gifts of nature to sustain life in extremely cold environments. This family of peptides, glycopeptides and proteins produced by diverse organisms including bacteria, yeast, insects and fish act by non-colligatively depressing the freezing temperature of the water below its melting point in a process termed thermal hysteresis which is then responsible for ice crystal equilibrium and inhibition of ice recrystallisation; the major cause of cell dehydration, membrane rupture and subsequent cryodamage. Scientists on the other hand have been exploring various substances as cryoprotectants. Some of the cryoprotectants in use include trehalose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), sucrose, propylene glycol (PG) and glycerol but their extensive application is limited mostly by toxicity, thus fueling the quest for better cryoprotectants. Hence, extracting or synthesizing antifreeze protein and testing their cryoprotective activity has become a popular topic among researchers. Research concerning AFPs encompasses lots of effort ranging from understanding their sources and mechanism of action, extraction and purification/synthesis to structural elucidation with the aim of achieving better outcomes in cryopreservation. This review explores the potential clinical application of AFPs in the cryopreservation of different cells, tissues and organs. Here, we discuss novel approaches, identify research gaps and propose future research directions in the application of AFPs based on recent studies with the aim of achieving successful clinical and commercial use of AFPs in the future.     

抗冻技术在改善冷冻水产品品质中的应用研究

阐述了抗冻技术的基本原理和在改善冷冻水产品品质上的应用现状，主要介绍了抗冻剂、漂洗工艺和漂洗剂的使用效果。说明了抗冻技术和漂洗工艺是保持冷冻水产品新鲜度、风味和防止其蛋白质冷冻变性的有效方法。为水产品的冷加工提供理论指导。     

Highly Concentrated Branched Oligosaccharides as Cryoprotectant for Surimi

Freeze-thawing studies at different concentrations, using an actomyosin solution (extracted from Alaska pollock), revealed that an 8% (w/v) solution of oligosaccharides mixture (HBOS) was most effective in cryoprotection. During frozen storage (-18°C), HBOS showed cryoprotective effects similar to sucrose and a sucrose+sorbitol mixture (1:1). Surimi gel prepared with HBOS showed higher hardness and more dense microstructure than others, although water holding capacity was slightly lower than the gel with sucrose+sorbitol. HBOS containing gel showed lower whiteness than sucrose but no difference with sucrose+sorbitol. HBOS appeared to have good potential as a non-sweet cryoprotectant of fish protein.     

Freeze-fracture electron microscopic and low temperature X-ray scattering studies of the effect of cryofixation upon serum low density lipoprotein structure

We report here a correlated X-ray diffraction and freeze-fracture electron microscope study of the effects of several cryofixation procedures upon human serum low density lipoprotein (LDL₂) structure. Only when the LDL₂ solutions contained 75%, by weight, glycerol were the room temperature and post cryofixation low temperature LDL₂ X-ray scattering curves indistinguishable from one another. Other cryofixation procedures, slow or rapid, with or without glycerol, resulted in differences between the room temperature and low temperature LDL₂ X-ray scattering curves, in part due to the effect of quenching upon the solvent. Freezeetching electron microscopy of the slowly cryofixed LDL₂ showed marked aggregation of the particles and an unusual morphological appearance. In contrast, after rapid cryofixation or cryofixation in the presence of glycerol, freeze-etch electron microscopy revealed well-isolated particles which had a knobby morphology. The results demonstrate that under certain conditions (in the presence of 75% glycerol) cryofixation results in minimal, if any, structural alteration of, at least, the LDL₂ lipid moiety. Further, this study underlines the more general conclusion that any high resolution structural study employing a cryofixation step must be interpreted with caution and the effect of cryofixation upon the sample structure need be evaluated by independent means.     

乳酸乳球菌乳脂亚种冷冻干燥保护剂优化及其贮藏稳定性

采用单因素试验、Plackett-Burman试验设计和Box-Behnken响应面法,筛选和优化乳酸乳球菌乳脂亚种KLDS4.0326真空冷冻干燥保护剂工艺,并分析冻干菌粉在不同温度下的贮藏稳定性。结果表明：脱脂乳粉、海藻糖和甘油含量是影响菌体细胞存活率的关键因素;以菌体细胞存活率为响应值,对3 个因素进行优化分析,保护剂最佳配比为：脱脂乳粉10.77 g/100 mL、海藻糖7.81 g/100 mL、甘油3.31 g/100 mL,此条件下菌体细胞存活率为（87.43±1.62）%,与理论预测值接近。在4 ℃和25 ℃条件下保藏12 个月后,菌体细胞存活率分别为40.63%和8.67%,冻干菌粉在4 ℃条件下具有更高贮藏稳定性。     

乳酸菌冷冻干燥保护剂的筛选

通过单因素比较和正交试验设计,确定适合保加利亚乳杆菌和嗜热链球菌的最佳冻干保护剂配方。通过对15种冻干保护剂单因素保护效果的比较,以及对选定的4种保护剂的正交试验结果,可以确定嗜热链球菌最佳冻干保护剂配方为脱脂乳粉12%、海藻糖1%、甘油3%、谷氨酸钠1%,保加利亚乳杆菌最佳冻干保护剂配方为脱脂乳粉8%、海藻糖1%、甘油2%、谷氨酸钠1.5%。     

Inhibition of frozen storage‐induced oxidation and structural changes in myofibril of common carp (Cyprinus carpio) surimi by cryoprotectant and hydrolysed whey protein addition

The objective of the present study was to evaluate the efficacy of combined cryoprotectants (sucrose + sorbitol) and whey protein isolate (WPI) hydrolysates to inhibit protein oxidation and quality loss in common carp (Cyprinus carpio) surimi during frozen storage at ?25 °C. With increasing storage time, the carbonyl content of myofibrillar proteins increased from 4.02 nmolmg^‐1 protein (0 day) to 7.25, 6.31, 5.26 and 4.83 nmol mg^?1 protein (180 days; P < 0.05) for the control and samples with cryoprotectants, with cryoprotectants + WPI hydrolysates and with cryoprotectants + propyl gallate, respectively; protein surface hydrophobicity and turbidity increased in a similar trend, while sulfhydryl content, Ca‐ATPase activity, protein solubility and protein thermal stability decreased (P < 0.05). These results suggest that treatments with combined cryoprotectants and antioxidative WPI hydrolysates offer an effective approach to reducing the extent of protein oxidation in common carp surimi, thereby limiting protein structural changes known to impair texture of surimi products.     

Polysaccharides can improve the survival of Lactiplantibacillus plantarum subjected to freeze-drying

Freeze-drying is one of the most commonly used methods of bacteria preservation. During this process, cryoprotectants can greatly reduce cellular damage. Micromolecular cryoprotectants have been widely adopted but have limited selectivity and protective effects. Therefore, explorations of other types of cryoprotectants are needed. This study aimed to explore the possibility of the macromolecular cryoprotectants and combinations of cryoprotectants to maintain bacterial activity. We found that the survival rate of Lactiplantibacillus plantarum AR113 after freeze-drying was 19% higher in the presence of soy polysaccharides than with trehalose, the best-performing micromolecular cryoprotectant. Moreover, a 90.52% survival rate of L. plantarum WCFS1 was achieved using the composite cryoprotectant containing soy polysaccharide and trehalose, which increased by 31.48 and 36.47% compared with adding solely trehalose or soy polysaccharide, respectively. These results demonstrate that macromolecular and micromolecular cryoprotectants have similar effects, and that combinations of macromolecular and micromolecular cryoprotectants have better protective effects. We further observed that the composite cryoprotectant can increase Lactobacilli survival by improving cell membrane integrity and lactate dehydrogenase activity. Our finding provides a new type of cryoprotectant that is safer and more effective, which can be extensively applied in the relevant food industry.     

直投式酸奶发酵剂的商品化生产研究

通过对不同保加利亚乳杆菌与嗜热链球菌菌株的酸化活力、黏度、蛋白水解能力和共生能力等生理学指标的测定,筛选出4对适于发酵生产用的菌株组合,并以菌株Y6+ST1作为进一步研究对象,获得其最佳增殖培养基为番茄汁为2.5%,乳糖为1.0%,酵母膏为0.5%,蛋白胨为1.5%,42℃培养6h后其活菌数达到1.4×109mL-1;Y6+ST1组合的最佳保护剂组成为脱脂奶粉为18.0%,甘油为2.0%,谷氨酸钠为1.0%和吐温-80为0.5%,经冷冻干燥后其活菌数达到3.62×1011g-1。该组合工业化生产的最佳工艺参数是培养温度42.3℃,pH值为6.4,搅拌转速86.8r/min和3%的接种量,1.0%补料(脱脂乳),发酵时间6h;-40℃,15h后,最终冷冻干燥产品活菌数为1011g-1。     

冻藏条件对蛤的蛋白质泠冻变性的影响及其改善的实验研究

以文蛤和波纹巴非蛤为对象研究了不同冻藏条件对肌原纤维Ca^2＋-ATPase活性、盐溶性蛋白溶解度的影响及在相同条件下添加抗冻剂的改善效果。结果表明，冻藏温度对贝肌肉、碎贝肉和贝糜变性有显著影响，且温度越低，变性越小；无完整细胞形态的贝糜的变性幅度最大，碎贝肉次之；添加抗冻剂能有效防止贝肉蛋白质冷冻变性，尤其能使贝糜肌原纤维蛋白质的稳定性大大提高。