抗冻蛋白(antifreeze proteins, AFPs)是一类能够在低温条件下抑制冰晶生长并调控冰晶形态的功能蛋白,在食品冷冻保鲜中具有应用潜力。该研究选取冬小麦(GaAFP)与银鳕鱼(DmAFP)来源的抗冻蛋白,通过毕赤酵母系统实现其异源表达,并评估其在冷冻保鲜中的应用。根据密码子偏好性合成基因,构建重组载体pPIC9K-GaAFP与pPICZαA-DmAFP并转化至毕赤酵母GS115与X-33菌株,经抗性筛选与发酵优化获得高产菌株,实现重组GaAFP与DmAFP高效表达。纯化后的重组蛋白表现出显著热滞活性。应用实验显示,AFPs处理的冷冻鸡胸肉解冻损失与蒸煮损失显著降低,持水性能显著提升;同时,AFPs处理后的马铃薯切片在冻融后水分分布较对照组更为均匀,细胞保水能力增强,且该性质与AFPs浓度相关。该研究为抗冻蛋白的规模化生产与产业化应用提供了技术参考。
Antifreeze proteins (AFPs) are functional proteins that inhibit ice crystal growth and modulate ice crystal morphology under low-temperature conditions, demonstrating significant application potential in the frozen preservation of food.This study focused on the heterologous expression of antifreeze proteins derived from winter wheat (GaAFP) and sablefish (DmAFP) using the Pichia pastoris system, and evaluated their efficacy in frozen preservation.Genes were synthesized based on codon preference, and recombinant vectors pPIC9K-GaAFP and pPICZαA-DmAFP were constructed and transformed into P.pastoris strains GS115 and X-33, respectively.High-yield strains were obtained through antibiotic resistance screening and fermentation optimization, leading to the efficient expression of recombinant GaAFP and DmAFP.The purified recombinant proteins exhibited significant thermal hysteresis activity.Application experiments demonstrated that frozen chicken breast treated with AFPs showed significantly reduced thawing loss and cooking loss, along with markedly improved water-holding capacity.Furthermore, potato slices treated with AFPs exhibited a more uniform water distribution after freeze-thaw cycles compared to the control group, indicating enhanced cellular water retention capacity, which was correlated with the AFP concentration.This study provided a technical reference for the large-scale production and industrial application of antifreeze proteins.
[1] 杨如意, 廖丽, 胥义. 抗冻蛋白冰结合位点特征及其研究综述[J]. 极地研究, 2025, 37(1):137-148.
YANG R Y, LIAO L, XU Y. Review of characteristics of Ice-binding sites of antifreeze proteins[J]. Chinese Journal of Polar Research, 2025, 37(1):137-148.
[2] MARSHALL C B, FLETCHER G L, DAVIES P L. Hyperactive antifreeze protein in a fish[J]. Nature, 2004, 429(6988):153.
[3] HEW C L, WANG N C, JOSHI S, et al. Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus[J]. Journal of Biological Chemistry, 1988, 263(24):12049-12055.
[4] GRAHAM L A, LIOU Y C, WALKER V K, et al. Hyperactive antifreeze protein from beetles[J]. Nature, 1997, 388(6644):727-728.
[5] GRIFFITH M, ALA P, YANG D S, et al. Antifreeze protein produced endogenously in winter rye leaves[J]. Plant Physiology, 1992, 100(2):593-596.
[6] MUÑOZ P A, MÁRQUEZ S L, GONZÁLEZ-NILO F D, et al. Structure and application of antifreeze proteins from Antarctic bacteria[J]. Microbial Cell Factories, 2017, 16(1):138.
[7] MA Q B, SHIBATA M, HAGIWARA T. Ice crystal recrystallization inhibition of type I antifreeze protein, type III antifreeze protein, and antifreeze glycoprotein: Effects of AF(G)Ps concentration and heat treatment[J]. Bioscience, Biotechnology, and Biochemistry, 2022, 86(5):635-645.
[8] 王彤, 陈生熬, 刘一萌, 等. 鱼类低温适应性机制与抗冻蛋白的研究进展[J]. 海洋渔业, 2024, 46(4):509-519.
WANG T, CHEN S A, LIU Y M, et al. Research progress on the mechanism of low temperature adaptation and antifreeze protein of fish[J]. Marine Fisheries, 2024, 46(4):509-519.
[9] OBADI M, XU B. Characteristics and applications of plant-derived antifreeze proteins in frozen dough: A review[J]. International Journal of Biological Macromolecules, 2024, 255:128202.
[10] HUANG Q, HU R Q, ZHU H, et al. Expression of multi-domain type III antifreeze proteins from the Antarctic eelpout (Lycodichths dearborni) in transgenic tobacco plants improves cold resistance[J]. Aquaculture and Fisheries, 2021, 6(2):186-191.
[11] ZIELKIEWICZ J. Mechanism of antifreeze protein functioning and the “anchored clathrate water” concept[J]. The Journal of Chemical Physics, 2023, 159(8):085101.
[12] CHOW-SHI-YÉE M, BRIARD J G, GRONDIN M, et al. Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells[J]. Protein Science, 2016, 25(5):974-986.
[13] ZHOU Y, JING K Y, JIA C K, et al. Effect of staged thawing assisted antifreeze protein on quality of repeatedly freeze-thawed minced pork[J]. Food Control, 2024, 159:110292.
[14] 肖书剑. 抗冻蛋白对兔肉品质特性及保鲜效果的影响研究[D]. 成都: 成都大学, 2025.
XIAO S J. Research on the effects of antifreeze proteins on the quality characteristics and preservation of rabbit meat[D]. Chengdu: Chengdu University, 2025.
[15] DU X, CHANG P, TIAN J Y, et al. Effect of ice structuring protein on the quality, thermal stability and oxidation of mirror carp (Cyprinus carpio L.) induced by freeze-thaw cycles[J]. LWT, 2020, 124:109140.
[16] WANG B, LI F F, PAN N, et al. Effect of ice structuring protein on the quality of quick-frozen patties subjected to multiple freeze-thaw cycles[J]. Meat Science, 2021, 172:108335.
[17] SUN X Y, LIU W Y, XIAO H W, et al. Effects of different freezing methods on the damage in potato tissues impregnated with trehalose[J]. LWT, 2024, 213:117096.
[18] 白艺朋. 热处理诱导青稞和鹰嘴豆籽粒结构和组分变化及其应用研究[D]. 无锡: 江南大学, 2022.
BAI Y P. Research on the changes in grain structure and components of barley and chickpea seeds induced by heat treatment and its application[D]. Wuxi: Jiangnan University, 2022.
[19] LING Z H, TRAN K C, ARNOLD J J, et al. Purification and characterization of recombinant human respiratory syncytial virus nonstructural protein NS1[J]. Protein Expression and Purification, 2008, 57(2):261-270.
[20] 战荣荣. 不同启动子调控的菊糖果糖转移酶在毕赤酵母中的高效分泌表达[D]. 无锡: 江南大学, 2014.
ZHAN R R. Efficient secretory expression of dexamethasone transferase regulated by different promoters in Pichia pastoris[D]. Wuxi: Jiangnan University, 2014.
[21] 张盛, 郝玉有, 储炬, 等. pH对毕赤酵母表达重组人复合α干扰素的降解影响[J]. 生物工程学报, 2008, 24(1):164-168.
ZHANG S, HAO Y Y, CHU J, et al. Effect of pH on proteolytic degradation of consensus interferon-α expressed by Pichia pastoris[J]. Chinese Journal of Biotechnology, 2008, 24(1):164-168.
[22] PAN L, CHEN X S, WANG K F, et al. Mechanisms of response to pH shock in microbial fermentation[J]. Bioprocess and Biosystems Engineering, 2020, 43(3):361-372.
[23] NIU C T, LIU G Z, YANG S J, et al. Enhanced expression of a novel trypsin from Streptomyces fradiae in Komagataella phaffii GS115 through combinational strategies of propeptide engineering and self-degredation sites modification[J]. International Journal of Biological Macromolecules, 2024, 254:127382.
[24] SHI Y Y, WANG H L, ZHENG Y, et al. Effects of ultrasound-assisted vacuum impregnation antifreeze protein on the water-holding capacity and texture properties of the yesso scallop adductor muscle during freeze-thaw cycles[J]. Foods, 2022, 11(3):320.
[25] CHEN J L, FAN Y Q, ZHANG X G, et al. Effect of antifreeze protein on the quality and microstructure of frozen chicken breasts[J]. Food Chemistry, 2023, 404:134555.
[26] 陈龙. 油炸过程中淀粉结构变化与吸油特性研究[D]. 无锡: 江南大学, 2019.
CHEN L. Research on the structural changes of starch and oil absorption characteristics during frying process[D]. Wuxi: Jiangnan University, 2019.
[27] LEYGONIE C, BRITZ T J, HOFFMAN L C. Impact of freezing and thawing on the quality of meat: Review[J]. Meat Science, 2012, 91(2):93-98.
