QIAO Ying,MA Rui,CHEN Zhigang.Effects of natural deep eutectic solvents on the viability of Streptococcus thermophilus during freeze-drying period[J].Journal of Nanjing Agricultural University,2018,41(5):931-938.[doi:10.7685/jnau.201711005]





Effects of natural deep eutectic solvents on the viability of Streptococcus thermophilus during freeze-drying period
乔颖 马锐 陈志刚
南京农业大学食品科技学院/糖组学与糖生物工程研究中心, 江苏 南京 210095
QIAO Ying MA Rui CHEN Zhigang
College of Food Science and Technology/Glycomics and Glycan Bioengineering Research Center, Nanjing Agricultural University, Nanjing 210095, China
natural deep eutectic solventsStreptococcus thermophilusfreeze-dryingcell lysiswater activity
[目的]以天然低共熔溶剂(NADES)作为低温保护剂,研究其在冷冻干燥期间对嗜热链球菌活力的影响。[方法]通过透射电子显微镜观察嗜热链球菌冷冻期间的损伤机制,并利用平板计数法测定嗜热链球菌的存活率。采用控制变量法分析嗜热链球菌细胞的最优裂解条件,测定嗜热链球菌中乳酸脱氢酶(LDH)和β-半乳糖苷酶活性,同时通过低场核磁共振仪测定NADES中的水分活度。[结果]嗜热链球菌冷冻后细胞中的细胞膜部分消失,细胞内的细胞质聚集成团,细胞间的链状结构消失,细胞表面蛋白完整。当体系中含有50% NADES时,嗜热链球菌的存活率明显高于体系中含有80% NADES的。在冷冻干燥后,只有氯化胆碱与山梨糖醇形成的NADES显著提高了嗜热链球菌冷冻干燥后的存活率(88.28%)。最优的裂解条件为:在1.2 mg·mL-1溶菌酶和0.5%(体积分数)TritonX-100的溶液中37℃裂解30 min。NADES对胞内LDH和β-半乳糖苷酶没有显著的保护作用。嗜热链球菌在含有氯化胆碱类的NADES中冷冻干燥的存活率较高。[结论]在冷冻期间细胞内冰晶对细胞膜和细胞质的损伤是主要的损伤机制,使用氯化胆碱和山梨糖醇形成的天然低共熔溶剂能显著提高嗜热链球菌冷冻干燥后的活力,嗜热链球菌的存活率与NADES中的水分活度有关。
[Objectives]In this paper,the important effects of natural deep eutectic solvents(NADES) as chemical protectants on Streptococcus thermophilus during freeze-drying were studied.[Methods]The damage mechanism was explored by transmission electron microscopy. The survival rate of S.thermophilus was determined by plate counting method. The control variables were used to explore the method of cell lysis. And lactate dehydrogenase(LDH) and β-galactosidase activities in S.thermophilus were determined. In addition,the experiment of water activity measurement was carried out by low-field nuclear magnetic resonance.[Results]The damage mechanism of S.thermophilus after freezing was studied,showing that the cell membrane disappeared,the intracellular cytoplasm agglomerated,the chain structure disappeared,and the cell surface protein was complete. The effects of different concentrations of NADES on S.thermophilus during pre-freezing period were different. It was found that the survival rate of S.thermophilus was significantly higher than that of 80% NADES,when 50% NADES was contained in the solvents. After freeze-drying,only NADES formed by choline chloride and D-sorbitol significantly increased the survival rate of S.thermophilus with a survival rate of 88.28%. The results showed that the best condition for cell lysis was the solution of 1.2 mg·mL-1 lysozyme and 0.5% TritonX-100 for 30 min at 37℃. NADES was found to have no significant protective effects on intracellular LDH and β-galactosidase. The survival rate of S.thermophilus was higher in NADES based on choline chloride after freeze-drying.[Conclusions]In the freezing period,intracellular ice crystals damage to the cell membrane and cytoplasm was the main damage mechanism. NADES formed by choline chloride and D-sorbitol significantly increased S.thermophilus viability after freeze-drying. The survival rate of S.thermophilus related to the changes of water activities in NADES.


[1] Smith E L,Abbott A P,Ryder K S. Deep eutectic solvents(DESs)and their applications[J]. Chemistry Review,2014,114(21):11060-11082.
[2] Abbott A P,Harris R C,Ryder K S,et al. Glycerol eutectics as sustainable solvent systems[J]. Green Chemistry,2011,13(1):82-90.
[3] Dimitrellou D,Kandylis P,Kourkoutas Y. Effect of cooling rate,freeze-drying,and storage on survival of free and immobilized Lactobacillus casei ATCC 393[J]. LWT-Food Science and Technology,2016,69:468-473.
[4] 李宝坤. 乳酸杆菌冷冻干燥生理损伤机制及保护策略的研究[D]. 无锡:江南大学,2011. Li B K. Study on physiological damage mechanism and protection strategy of Lactobacillus freeze-drying[D]. Wuxi:Jiangnan University,2011(in Chinese with English abstract).
[5] Romano N,Schebor C,Mobili P,et al. Role of mono-and oligosaccharides from FOS as stabilizing agents during freeze-drying and storage of Lactobacillus delbrueckii subsp. bulgaricus[J]. Food Research International,2016,90:251-258.
[6] Santivarangkna C,Wenning M,Foerst P,et al. Damage of cell envelope of Lactobacillus helveticus during vacuum drying[J]. Journal of Applied Microbiology,2007,102(3):748-756.
[7] Halim M,Anizawanie N,Mustafa M,et al. Effect of encapsulant and cryoprotectant on the viability of probiotic Pediococcus acidilactici ATCC 8042 during freeze-drying and exposure to high acidity,bile salts and heat[J]. LWT-Food Science and Technology,2017,81:210-216.
[8] Li B K,Tian F,Liu X,et al. Effects of cryoprotectants on viability of Lactobacillus reuteri CICC6226[J]. Applied Microbiology Biotechnology,2011,92(3):609-616.
[9] Durand E,Lecomte J,Bar A B,et al. Evaluation of deep eutectic solvent-water binary mixtures for lipase-catalyzed lipophilization of phenolic acids[J]. Green Chemistry,2013,15(8):2275-2282.
[10] Gutiérrez M C,Ferrer M L,Yuste L,et al. Bacteria incorporation in deep-eutectic solvents through freeze-drying[J]. Angewandte Chemie International Edition,2010,49(12):2158-2162.
[11] Huang Y,Feng F,Jiang J,et al. Green and efficient extraction of rutin from tartary buckwheat hull by using natural deep eutectic solvents[J]. Food Chemistry,2017,221:1400-1405.
[12] Hajmeer M,Ceylane E,Marsden J L,et al. Impact of sodium chloride on Escherichia coli O157:H7 and Staphylococcus aureus analysed using transmission electron microscopy[J]. Food Microbiology,2006,23(5):446-452.
[13] Malanovic N,Lohner K. Gram-positive bacterial cell envelopes:the impact on the activity of antimicrobial peptides[J]. Biochimica et Biophysica Acta(BBA):Biomembranes,2016,1858(5):936-946.
[14] Babu V,Choudhury B. Methods of cell lysis and effect of detergents for the recovery of nitrile metabolizing enzyme from Amycolatopsis sp. ⅡTR215[J]. Journal of Genetic Engineering and Biotechnology,2013,11(2):117-122.
[15] Qiao Y,Huang Y,Feng F,et al. Efficient enzymatic synthesis and antibacterial activity of andrographolide glycoside[J]. Process Biochemistry,2016,51(5):675-680.
[16] Li T,Rui X,Wang K,et al. Study of the dynamic states of water and effects of high-pressure homogenization on water distribution in tofu by using low-field nuclear magnetic resonance[J]. Innovative Food Science & Emerging Technologies,2015,30:61-68.
[17] King V E,Su J. Dehydration of Lactobacillus acidophilus[J]. Process Biochemistry,1993,28(1):47-52.
[18] Meng J,Zhu X,Gao S M,et al. Characterization of surface layer proteins and its role in probiotic properties of three Lactobacillus strains[J]. Int J Biol Macromol,2014,65:110-114.
[19] Han B,Bischof J C. Direct cell injury associated with eutectic crystallization during freezing[J]. Cryobiology,2004,48(1):8-21.
[20] Hub L Z. Protectants used in the cryopreservation of microorganisms[J]. Cryobiology,2003,46(3):205-229.
[21] Sanchezfernandez A,Edler K J,Arnold T,et al. Protein conformation in pure and hydrated deep eutectic solvents[J]. Physical Chemistry Chemical Physics,2017,19(13):8667-8670.
[22] Zeng Q,Wang Y,Huang Y,et al. Deep eutectic solvents as novel extraction media for protein partitioning[J]. Analyst,2014,139(10):2565-2573.
[23] Tang B,Row K H. Recent developments in deep eutectic solvents in chemical sciences[J]. Monatshefte für Chemie-Chemical Monthly,2013,144(10):1427-1454.
[24] Xiao Q,Lim L T,Zhou Y,et al. Drying process of pullulan edible films forming solutions studied by low-field NMR[J]. Food Chemistry,2017,230:611-617.
[25] Zhang L,Qu M,Yao J,et al. Effect of high hydrostatic pressure on the viability of Streptococcus thermophilus bacteriophages isolated from cheese[J]. Innovative Food Science & Emerging Technologies,2015,29:113-118.
[26] 高薇,韩雪,张兰威. 乳酸菌渗透胁迫相关相容性溶质及其转运机制研究进展[J]. 微生物学通报,2013,40(11):2097-2106. Gao W,Han X,Zhang L W. Advances in research on compatibility solute and its transport mechanism of lactic acid bacterial osmotic stress[J]. Microbiology Bulletin,2013,40(11):2097-2106(in Chinese with English abstract).


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更新日期/Last Update: 1900-01-01