微生物学报

Methods	Strains	Term	Results	Mechanisms	References
Heat	Enterococcus faecium	52 ℃ 15 min	Increased tolerance to H₂O₂, ethanol, acid, and alkaline stresses		[4]
	Tetragenococcus halophilus	45 ℃ 1.5 h	Seven-fold increase in survival in ethanol-stressed environments	Involvement in energy metabolism and upregulation of membrane transporter protein genes	[87]
	Lactobacillus acidophilus	65 ℃ 40 min	Increased stability when stored at 37 ℃ and 42 ℃ for one week		[15]
	Lactobacillus acidophilus	45 ℃ 30 min	Freeze-drying survival rate increased from 39.1% to 56.3%	Changes in key enzymes of glycolysis and Na⁺-K⁺ ATPase and increased galactose production	[88]
Cold	Streptococcus thermophilus	10 ℃ 2 h	Increased tolerance to gastric fluids		[89]
	Lactobacillus delbrueckii ssp. bulgaricus	10 ℃ 2 h	Lyophilisation survival rate increased by 16.06%	Expression of two cold shock-induced genes and six heat shock-induced genes were upregulated	[79]
	Lactobacillus kefiranofaciens	20 ℃ 1 h	Inducing cross-stress and increasing resistance to other adversarial environments	Up-regulation of the expression of the molecular chaperones DnaK and GroEl	[90]
	Lactobacillus helveticus	10 ℃ 2 h		Increased synthesis of cyclopropane fatty acids and enhanced cell membrane fluidity	[91]
	Lactobacillus brevis	-5 ℃ 2 h	Extended shelf life	Production of surface proteins	[92]
Acid	Lactobacillus casei	pH 4.510 min	100-fold increase in survival at pH 2.5	Intracellular malate and histidine accumulation	[81]
	Lactobacillus plantarum	pH 3.0 1 h	Increased survival rate after 180 days of storage at room temperature		[93]
	Lactobacillus delbrueckii ssp. bulgaricus	pH 5.0 MRS	Freeze-drying survival rate increased to 68.3%		[79]
	Lactobacillus plantarum	pH 4.5 2 h	Increased lyophilisation survival	Genes involved in fatty acid synthesis and amino acid metabolism and sugar metabolism were significantly upregulated	[94]
NaCl	Lactobacillus acidophilus	0.6 mol/L NaCl		Increased synthesis of surface proteins	[95]
	Lactobacillus plantarum	0.4 mol/L NaCl	Increased lyophilisation survival	Up-regulation of K⁺ transporter-related trkA gene increases cell membrane unsaturated fatty acid content	[96]
	Lactobacillus delbrueckii ssp. bulgaricus	0.2 mol/L NaCl		Increased accumulation of the compatible solute glycine betaine	[97]
	Lactobacillus delbrueckii ssp.bulgaricus	2% NaCl 2 h	Increased glucose utilisation	Increased activity of glycolytic enzymes	[98]
H₂O₂	Bifidobacterium animalis	1.5 mmol/L H₂O₂		Up-regulation of genes encoding thioredoxin systems and divalent cation transporter protein genes	[99]
	Lactobacillus rhamnosus	0.5 mmol/L H₂O₂	Increased survival in sublethal conditions	Enhanced regulation of amino acid metabolism and group sensing pathways	[100]
	Bifidobacterium	210 ppm dissolved oxygen	No loss of viability after 35 days of storage		[101]

Methods	Strains	Term	Results	Mechanisms	References
Heat	Enterococcus faecium	52 ℃ 15 min	Increased tolerance to H₂O₂, ethanol, acid, and alkaline stresses		[4]
	Tetragenococcus halophilus	45 ℃ 1.5 h	Seven-fold increase in survival in ethanol-stressed environments	Involvement in energy metabolism and upregulation of membrane transporter protein genes	[87]
	Lactobacillus acidophilus	65 ℃ 40 min	Increased stability when stored at 37 ℃ and 42 ℃ for one week		[15]
	Lactobacillus acidophilus	45 ℃ 30 min	Freeze-drying survival rate increased from 39.1% to 56.3%	Changes in key enzymes of glycolysis and Na⁺-K⁺ ATPase and increased galactose production	[88]
Cold	Streptococcus thermophilus	10 ℃ 2 h	Increased tolerance to gastric fluids		[89]
	Lactobacillus delbrueckii ssp. bulgaricus	10 ℃ 2 h	Lyophilisation survival rate increased by 16.06%	Expression of two cold shock-induced genes and six heat shock-induced genes were upregulated	[79]
	Lactobacillus kefiranofaciens	20 ℃ 1 h	Inducing cross-stress and increasing resistance to other adversarial environments	Up-regulation of the expression of the molecular chaperones DnaK and GroEl	[90]
	Lactobacillus helveticus	10 ℃ 2 h		Increased synthesis of cyclopropane fatty acids and enhanced cell membrane fluidity	[91]
	Lactobacillus brevis	-5 ℃ 2 h	Extended shelf life	Production of surface proteins	[92]
Acid	Lactobacillus casei	pH 4.510 min	100-fold increase in survival at pH 2.5	Intracellular malate and histidine accumulation	[81]
	Lactobacillus plantarum	pH 3.0 1 h	Increased survival rate after 180 days of storage at room temperature		[93]
	Lactobacillus delbrueckii ssp. bulgaricus	pH 5.0 MRS	Freeze-drying survival rate increased to 68.3%		[79]
	Lactobacillus plantarum	pH 4.5 2 h	Increased lyophilisation survival	Genes involved in fatty acid synthesis and amino acid metabolism and sugar metabolism were significantly upregulated	[94]
NaCl	Lactobacillus acidophilus	0.6 mol/L NaCl		Increased synthesis of surface proteins	[95]
	Lactobacillus plantarum	0.4 mol/L NaCl	Increased lyophilisation survival	Up-regulation of K⁺ transporter-related trkA gene increases cell membrane unsaturated fatty acid content	[96]
	Lactobacillus delbrueckii ssp. bulgaricus	0.2 mol/L NaCl		Increased accumulation of the compatible solute glycine betaine	[97]
	Lactobacillus delbrueckii ssp.bulgaricus	2% NaCl 2 h	Increased glucose utilisation	Increased activity of glycolytic enzymes	[98]
H₂O₂	Bifidobacterium animalis	1.5 mmol/L H₂O₂		Up-regulation of genes encoding thioredoxin systems and divalent cation transporter protein genes	[99]
	Lactobacillus rhamnosus	0.5 mmol/L H₂O₂	Increased survival in sublethal conditions	Enhanced regulation of amino acid metabolism and group sensing pathways	[100]
	Bifidobacterium	210 ppm dissolved oxygen	No loss of viability after 35 days of storage		[101]

乳酸菌在不同胁迫下应激反应及高活性保护机制研究进展

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陈宁 ¹^,² , 吴磊 ² , 谢新强 ² , 赵昕宇 ³^,^* , 吴清平 ²^,^*

微生物学报 | 综述 2025,65(2): 421-436

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微生物学报 | 综述 2025, 65(2): 421-436

乳酸菌在不同胁迫下应激反应及高活性保护机制研究进展

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陈宁¹^,², 吴磊², 谢新强², 赵昕宇³^,^*, 吴清平²^,^*

作者信息

1 华南农业大学食品学院，广东广州

2 广东省科学院微生物研究所，华南应用微生物国家重点实验室，广东省微生物安全与健康重点实验室，国家卫健委微生物食品营养与安全科技创新平台，广东广州

3 广东科环生物科技有限公司，广东广州

Research progress in responses and high activity protection mechanisms of lactic acid bacteria under different stresses

Ning CHEN¹^,², Lei WU², Xinqiang XIE², Xinyu ZHAO³^,^*, Qingping WU²^,^*

Affiliations

1 College of Food Science, South China Agricultural University, Guangzhou, Guangdong, China

2 State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Safety and Health, National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, Guangdong, China

3 Guangdong Kehuan Biotechnology Co. , Ltd. , Guangzhou, Guangdong, China

出版时间: 2025-02-04 doi: 10.13343/j.cnki.wsxb.20240596

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摘要

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乳酸菌在食品和医学领域应用广阔，在食品发酵行业中可用作发酵剂和功能性菌粉。乳酸菌的活性和作用效果会受到各种环境压力的影响，如渗透压、温度、氧、酸、胆盐等。可食用的乳酸菌能够发挥多种多样的健康疗效，但其活菌数在生产、储藏以及人体消化过程中均有减少。因此，本文系统阐述了乳酸菌在其生产、储藏和消化过程中遭受的不同胁迫环境以及面对环境压力的应激反应，并从隔离胁迫环境和增加菌株抗逆性2个方面总结了现有高活性保护策略与机制，以期为乳酸菌菌株的选育与产品开发应用提供必要的理论依据。

关键词

乳酸菌 / 胁迫 / 应激反应 / 保护机制

Abstract

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Lactic acid bacteria (LAB) have extensive applications in food and medicine fields. They are used as starters and functional probiotics in food fermentation. The activity and performance of LAB are influenced by various environmental stresses involving osmotic pressure, temperature, oxygen, acidity, and bile salts. Edible LAB can provide numerous health benefits. However, their viable counts decrease during production, storage, and digestion. This paper systematically discusses the different stressful environments faced by LAB during production, storage, and digestion, as well as their stress responses. Furthermore, this paper summarizes existing high activity protection strategies and mechanisms from two aspects: isolating from stressful environments and enhancing strain resistance. This review aims to provide theoretical support for LAB strain engineering and product development.

Key words

lactic acid bacteria / stress / stress response / protection mechanism

引用本文

陈宁, 吴磊, 谢新强, 赵昕宇, 吴清平. 乳酸菌在不同胁迫下应激反应及高活性保护机制研究进展. 微生物学报, 2025 , 65 (2) : 421 -436 . DOI: 10.13343/j.cnki.wsxb.20240596

Ning CHEN, Lei WU, Xinqiang XIE, Xinyu ZHAO, Qingping WU. Research progress in responses and high activity protection mechanisms of lactic acid bacteria under different stresses[J]. Acta Microbiologica Sinica, 2025 , 65 (2) : 421 -436 . DOI: 10.13343/j.cnki.wsxb.20240596

正文

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乳酸菌(lactic acid bacteria, LAB)是一类无芽孢的革兰氏阳性菌株，它们通过发酵产生大量活性物质，这些物质有助于促进健康，被广泛应用于发酵食品中^[1]。在食品工业中广泛使用的LAB主要有链球菌属(Streptococcus)、双歧杆菌属(Bifidobacterium)、乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)、酒球菌属(Oenococcus)和魏斯氏菌属(Weissella)等^[2]。LAB可以调节肠道菌群^[3]、降低胆固醇、改善睡眠以及提高人体免疫力^[4]。本团队已挖掘出多株有自主知识产权且具有良好益生功能的LAB^[5-7]，其中Wu等^[8]从百岁老人肠道微生物中筛选出一株可以产抗坏血酸的具有抗氧化功能的植物乳杆菌，Yang等^[9]通过动物实验表明，副干酪乳杆菌可以缓解大鼠的高胆固醇血症，还有一株高产γ-氨基丁酸的植物乳杆菌能够提高睡眠障碍人群的睡眠质量^[10]。LAB的生物转化产品在食品工业中得到了广泛应用，以功能性食品的形式食用可以改善人体健康并有助于预防疾病^[11]。然而，LAB在食品发酵和生产过程中遇到了各种非生物和生物胁迫，严重影响其工业化生产效率和功能活性的发挥^[2]。LAB在不同环节与用途所遭受的胁迫也不尽相同。在用作发酵剂时，LAB必须在生产和储存过程中抵抗离心、干燥等工艺程序带来的胁迫，而当用作活疫苗或益生菌菌粉时，LAB则需要承受胃肠道的压力^[12]。隔离外界胁迫是工业上保护LAB活性最高效与经济的方法之一。Chen等^[13]发现重组抗冻肽可以抑制嗜热链球菌细胞凋亡，提高其冷冻胁迫下存活率。Liu等^[14]设计出具有pH响应和黏接性能的硫代氧化魔芋葡甘露聚糖微球，可以使LAB免受胃酸的伤害。除此之外，还有许多研究证明，LAB为了应对不同类型的压力自身也开发了多种保护策略来应对外界的恶劣条件和环境的快速变化^[15-17]。通过合成分子伴侣、酶、抗氧化剂和胞外多糖等特殊的生物分子，可以增强LAB对外界环境胁迫的耐受性^[18]，并且能够在显著提高自身于应激条件下的耐受性的同时不会损害其生理活性^[19]。因此，如何提高LAB的抗胁迫能力成为解决生产问题的关键。

为扩大益生菌市场，满足消费者的需求，迫切需要开发和制备具有益生功能的LAB产品。环境胁迫不仅对于LAB工业应用至关重要，而且会影响到菌株的生理活性及其益生功能。因此，本综述旨在总结不同环境胁迫下LAB的应激反应及其分子调控机制，从隔离胁迫环境和增加菌株抗逆性2个方面总结了现有高活性保护策略与机制，对LAB产品菌种的筛选和改良，以及生产工艺优化都具有重要指导意义。

1 LAB受到的胁迫环境

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LAB产品从制备到消费，需历经多重环境考验，图1总结了LAB在生产、储藏、消化3个环节所面临的胁迫因素。

1.1 乳酸菌生产和储藏中受到的胁迫

1.1.1 乳酸胁迫

LAB在高密度培养时会产生大量乳酸，很大程度上抑制了LAB的生长活力，是工业生产上无法避免的压力环境，其含量会随着LAB生长阶段的变化而变化^[20]。乳酸会通过被动扩散进入细胞膜并解离成质子和酸残基，一旦它们进入细胞，则无法通过细胞膜返回外部，导致质子在细胞内积累，从而影响跨膜电位，进而影响多种跨膜转运机制的能量来源^[2]。Zhu等^[21]发现乳酸乳球菌中参与嘧啶生物合成途径和甘氨酸或甜菜碱转运过程的基因也会在酸胁迫期间上调。若在LAB对数生长阶段用较温和的酸性条件刺激，它们能够展现出启动适应性反应的能力，这一过程被称为酸耐受应激(acid tolerance response, ATR)^[12]。例如，将LAB短暂地暴露于pH 5.0的环境中1 h，再转移至pH 2.0的环境中，其活菌数量并未降低^[22]。除上述适应性反应外，LAB还能够利用ATP酶(也称H⁺-ATP酶)来维持细胞内pH稳态，它通过水解ATP将H⁺泵出细胞，或利用ATP水解产生的能量从细胞中去除H⁺，在维持pH稳态中发挥重要作用^[23]。除此之外，LAB还可以通过氨基酸的脱羧反应，从细胞中吸收H⁺，然后通过细胞膜排出，使细胞内pH值升高^[12]。

LAB菌株生长过程中的乳酸环境较为常见，了解酸应激反应的分子机制有助于调整和改造这些LAB菌株用于工业应用。

1.1.2 渗透压胁迫

针对1.1.1所述的乳酸胁迫，工业上一般会采取流加碱液的方式来中和乳酸对菌体生长的反馈抑制，然而这种方法伴随大量盐分的产生，进而导致渗透压升高^[20]。渗透压的提升是影响许多发酵食品生产的一个重要问题。首先，LAB细胞的膜蛋白可直接或间接调节细胞膜对盐离子的渗透性，Na⁺/H⁺逆向转运蛋白会外排Na⁺，这不仅能减轻Na⁺对菌体细胞的毒害作用，而且能通过增加对K⁺的吸收来恢复Na⁺/K⁺比^[24]。为了更好地应对渗透压的变化，LAB还会积累更多的相容性物质^[25]，以此来平衡细胞内外渗透压，提高细胞的耐受阈值^[12]，并减少细胞失水的风险。相容性溶质是Brown等^[26]在1972年首次提出的可以在细胞中以高浓度聚集，而不妨碍细胞功能的蛋白质分子正确折叠的小分子或结构化合物^[27]。目前报道的参与渗透胁迫应激的相容性物质有甘氨酸甜菜碱和胆碱等^[28]。然而，不同种类的LAB在相容性分子的积累机制上也存在差异^[29-30]。在植物乳杆菌中，渗透剂的吸收是通过对甜菜碱具有高亲和力并能够转运季铵盐化合物的QacT系统协调的^[31]。相比之下，在乳酸乳球菌中，甜菜碱和脯氨酸的摄取是由opuA/busA操纵子控制，该操纵子编码ABC家族转运蛋白，通过消耗2个ATP分子来转运单个甜菜碱分子，转运蛋白的活性取决于细胞内离子强度的变化以及转运蛋白本身与细胞膜之间的静电作用^[32]。

综上所述，LAB在面对外界渗透压的改变时，一是依靠细胞膜对膜电位的维持，二是通过积累相容性溶质来适应。

1.1.3 温度胁迫

LAB产品的细胞数量和活力是影响产品质量的关键因素^[33]。冷冻干燥技术目前被认为是保持菌株产品活性的最佳干燥方式之一^[34]。与其他干燥方式相比，冷冻干燥有很多优点，但同样地，由于细胞暴露于极冷环境，它也可能对细胞壁、细胞膜和DNA造成生理损伤，从而影响细胞的活力和功能活性^[35]。LAB细胞会诱导产生冷诱导蛋白减轻冷刺激造成的细胞损伤，该蛋白能调控低温胁迫下的细胞来保证正常DNA的超螺旋结构和增加膜质中短链或不饱和脂肪酸的比例，以维持细胞膜的流动性。尽管冷冻干燥可以长期保持LAB细胞的活力，但由于其成本高昂，在乳品行业中，喷雾干燥仍然是普遍的干燥方式，LAB最适的生长温度在30-37 ℃之间^[36]，而喷雾干燥过程中的高温会引起热应激，破坏细胞稳态，导致细胞中蛋白质的变性和聚集，以及影响DNA等生物大分子的稳定性和膜的流动性^[37]。公丕民^[38]的研究发现，喷雾干燥过程中细胞膜通透性的改变是导致细胞死亡的主要原因。不仅细胞膜脂肪酸的组成在干燥之后会发生相变，细胞壁的修复功能也会受到抑制。对于商业益生菌的生产而言，随后的储存环境中的温度也是能否保证其活菌数量的关键条件，LAB的短期保存以4 ℃储藏为主，而长期储藏则以-20 ℃效果最佳^[35]。Shu等^[39]的研究发现，由于常温下的菌粉胞内水分活度较高，而低温储藏细胞生长代谢较慢，因此常温储藏会更快丧失活力。此外，研究表明，当储藏温度高于玻璃化转变温度时，LAB产品会呈现橡胶态，导致接触时间减少，从而更容易发生结块现象^[40]。

因此，研究冷应激以及热应激可以充分了解LAB对不同应激条件的适应性，是之后开发相应保护策略来保证在食品生产和储藏环境中质量和功能的重要一步。

1.1.4 氧胁迫

LAB最终产品的总活菌数量与活力对产品的质量与益生功效至关重要，储藏环境的高氧气含量不仅会降低菌株的酶活性，还会促进不饱和脂肪酸的氧化，此外，LAB在生长代谢过程中产生的活性氧(reactive oxygen species, ROS)也会对菌体的蛋白质和DNA造成较大损伤，从而降低细胞活性^[41]。活性氧广泛指氧来源的自由基和非自由基，由于它们含有不成对的电子，因此具有很高的化学反应活性，例如超氧阴离子(O^2-)、臭氧(O₃)和单线态氧(1O₂)等，自然条件下，ROS来源于胞内酶的自氧化、环境中的氧化还原反应、竞争性微生物的释放等^[42]。LAB与氧胁迫应激反应相关的酶主要有NADH-氧化酶/NADH-过氧化物酶和超氧化物歧化酶(superoxide dismutase, SOD)，NADH-氧化酶/NADH-过氧化物酶系统可在NADH氧化酶作用下生成H₂O₂，再通过NADH过氧化物酶将其分解为水，防止LAB细胞中氧气积聚^[2]。Gibson等^[43]通过插入失活编码NADH-氧化酶的基因来构建NADH-氧化酶缺陷突变体，证明了其在碳水化合物和氧含量高的环境中至关重要。SOD是LAB中最重要的抗氧化酶之一，它能使O^2-突变，从而降低细胞内游离金属阳离子的浓度，并有效减轻H₂O₂对细胞造成的损伤^[44]。除了从抗氧化角度抵抗氧胁迫之外，维持细胞内的还原性也同样重要。研究表明，在添加谷胱甘肽的培养基中生长的LAB细胞，对H₂O₂处理的抗性显著增加，这可能是因为添加谷胱甘肽之后激活了细胞中的谷胱甘肽-谷胱甘肽过氧化物酶-谷胱甘肽还原酶系统^[45]。Jänsch等^[46]也利用基因敲除的方法证明了gshR基因敲除株与野生型菌株相比，前者在改良的MRS培养基上的有氧生长速率显著降低。

综上所述，LAB所受到的氧胁迫不仅来源于外界分子氧的攻击，其本身的菌株生长代谢过程中产生的活性氧也是一个不可忽视的重要来源。在储藏过程中，LAB产品活性的降低不仅归因于氧的胁迫，储藏环境温度的改变也是一个至关重要的影响因素。因此，许多研究也着重于探究菌株的交叉保护机制，即探讨如何通过轻微胁迫下的预适应来提高LAB对其他环境胁迫的耐受性^[2]。

1.2 消化过程中的胁迫

1.2.1 胃酸胁迫

消化道是抵抗外来病原微生物对机体损害的天然屏障，然而具有益生作用的LAB产品同样需要通过这一屏障来发挥作用。LAB所面临的酸胁迫环境主要有两方面，一是在增殖过程中所产生的乳酸会影响菌体自身的生长和代谢，二是pH值接近2.0的胃酸^[12]。胃酸是LAB进入人体后发挥益生作用所遇到的第一个胁迫环境，LAB的益生作用的发挥取决于通过胃液之后所到达作用部位的活菌数量^[47]。与菌株生长代谢产生的乳酸不同，胃液的独特成分和低pH值对于LAB的生存来说是一个巨大的挑战，这归因于胃的酸性条件会在细胞水平上引起严重的形态和表型改变，例如微生物细胞膜组成的变化、DNA的损伤以及肽聚糖成分的改变^[48]。细胞膜是大多数细胞溶质交换的主要屏障，同时也是应激损伤的主要部位，对于维持细胞内外pH稳态至关重要。为了防止质子流入细胞，细胞通常会通过调节脂肪酸的组成来改变膜的流动性，例如在双歧杆菌中，细胞膜上较高比例的环丙烷脂肪酸与较长的脂肪酸链有助于降低膜的流动性，从而增强菌株的耐酸能力^[49]。Wei等^[50]也发现，在低pH条件下，长双歧杆菌会通过重塑肽聚糖生物合成和脂肪酸代谢来修饰其细胞壁。另外，当LAB的DNA暴露于强酸中时，由于碱基的质子化以及糖苷键的断裂，会产生无嘌呤和无嘧啶位点^[12]。

LAB在遭受胃酸胁迫时，主要是通过加强细胞屏障防止外来质子流入造成细胞内外pH失衡。

1.2.2 胆盐胁迫

胆盐是胆汁中参与消化吸收脂肪的主要成分^[51]，它通过溶解细胞膜中的磷脂和膜蛋白来达到杀菌的目的^[52]。LAB需要经过多重消化道之后才能到达肠道发挥益生作用，而消化道的上一级环境引起的LAB代谢变化可能会影响下一级环境中的生理功能^[53]。为了维持自身稳态，LAB细胞会通过调控膜蛋白的基因表达来控制外来胆盐的渗透率，在嗜热链球菌中被证明通过调控基因来保持细胞膜完整性从而增加对胆盐的抗性^[54]。与此同时，LAB为了克服胆盐胁迫，通过调整其能量代谢的途径、水平来维持稳态^[2]。Sanchez等^[55]对长双歧杆菌的蛋白质组学与转录组学进行分析后发现，在胆盐胁迫下，参与转录和翻译以及糖酵解和丙酮酸分解代谢的酶表达量增加。乌日娜^[56]也发现，干酪乳杆菌在含有1.5%胆盐的培养基中生长后，其应激蛋白、糖代谢蛋白等26种蛋白质的表达发生了变化。当受到胆盐刺激时，LAB会产生能够将结合型胆盐转化成游离型胆盐的胆盐水解酶(bile salt hydrolase, BSH)，来减少胆盐对细胞的毒害^[51]。有研究将不含BSH与含BSH的LAB比较发现，含有BSH的LAB对共轭胆汁酸有更强的抵抗力^[57]。Grill等^[58]也发现，无共轭胆汁盐水解酶活性的突变株的生长速率受胆盐的影响更大。上述结论表明，LAB在面对胆盐胁迫时具有多种适应性机制和策略。

2 乳酸菌在不同胁迫环境下的应激保护策略及保护机制

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不同环境对LAB的胁迫有所不同，从对恶劣环境进行隔离和增强菌株自身抗逆性2个方面来设计不同的保护策略能够具有针对性和可靠性。图2总结了目前2个方面的应激保护策略。

2.1 隔离胁迫环境

2.1.1 添加保护剂

LAB通常以菌粉的形式用作膳食补充剂^[59]、奶酪或酸奶生产的发酵剂。然而，LAB发酵剂很难避免冷冻和干燥过程中的各种损害。因此，可以选择添加合适的保护剂来提高LAB在生产时的存活率和活力^[60]。吴家琳等总结了常用的冻干保护剂有糖类、蛋白质类、盐类、高分子类等^[61]，其中糖类保护剂研究最为广泛，特别是非还原性二糖，例如海藻糖和蔗糖，这归因于它们较高的玻璃化转变温度更适于低温保存^[62]。Ge等^[63]的研究也佐证了由于玻璃化转变温度的提高，有效地减轻了在冻干过程中由冰晶引起的机械损伤，并保持细胞膜上Na⁺/K⁺-ATP酶的活性。海藻糖、蔗糖和脱脂乳的组合冷冻干燥后的存活率在83%-85%之间^[64]。蛋白质类也是冻干保护剂的重要一部分，Ying等^[65]发现乳清蛋白的基质缓冲能力可以让LAB在极端的pH环境中保持活力。除一些常见物质之外，一些氨基酸物质则能够结合细胞内的蛋白质结构中的氨基基团，从而稳定蛋白质的功能^[66]，并且能够调节再水化过程中的Zeta电位，恢复表面电荷后从而提高对Caco-2细胞系的黏附能力，使得菌株更好地存活于胃肠道环境^[67]。除使用单一的保护剂之外，研究人员也探究了不同种类保护剂的复配方式。Savedboworn等^[68]通过将大米蛋白和低聚果糖组合作为植物乳杆菌的冻干保护剂，其能够保持细胞表面高度的疏水性，最后冻干后的菌粉与活菌对食源性致病菌的抑制能力无差异。

Zhou等^[69]研究发现，l-谷氨酸钠是对具有促睡眠功能的植物乳杆菌L-5的高效冻干保护剂，通过调控丙酮酸生物合成和代谢等过程来提高菌株对真空和冷应激的响应能力，活菌数6个月后仍稳定在4.80×10¹¹ CFU/g。

2.1.2 微囊化

微囊化技术是指利用天然或合成的高分子材料作为壁材，将固体、液体等芯材包埋在生物相容性聚合物基质或壳体内的一类技术，微囊化已被证明是保护LAB免遭上消化道降解的一项有效手段^[70]。有研究报道微胶囊化可保护益生菌免受胃液侵蚀，提高其在胃肠道中的存活率^[71]。刘会平等^[72]利用海藻酸钠和氯化钙离子交换反应对干酪乳杆菌进行双层包埋，在2-6 ℃下干燥36-72 h后，所得的微胶囊具有较强的耐酸耐胆盐能力与储藏稳定性。除此之外，微囊化也是抵抗加工过程环境胁迫的有效方式。Sun等^[60]通过干燥动力学实验发现，海藻糖-乳清蛋白-支链淀粉凝胶体系具有优异的持水性，这归因于海藻糖降低了水凝胶的储能模量，减弱了蛋白质和多糖之间的疏水相互作用、二硫键和氢键，从而保证了植物乳杆菌生存的最低水分需求，并增强了其在冷冻干燥过程中的活力。根据LAB的菌株特异性设计不同的包埋保护方式，不仅能够提高在消化道和储藏时的稳定性，而且能够达到缓释的目的^[73]。Eratte等^[74]将干酪乳杆菌与金枪鱼油共包埋于乳清蛋白分离物——阿拉伯胶络合物中，体外模拟消化发现，由于总omega-3脂肪酸释放量的提高，使得微胶囊在通过唾液、胃液之后干酪乳杆菌还有较高的存活率，并提高了黏附在肠壁上的能力。

现有的微囊化的方式仍有许多不足，高成本以及复杂的工艺设计限制了大部分微囊化的工业应用，其次，LAB微囊化的粒径、形态不能满足所有产品的需求。因此，如何根据菌株的特异性与LAB产品应用场景而设计不同的微囊化方法是一个值得深入研究的领域。

2.2 增强菌株抗逆性

除在外部隔离胁迫环境之外，增强菌株本身的抗逆性更为重要。目前相关的研究方法主要可分为改变培养条件、预适应、基因工程三类。图3所示为增强菌株抗逆性的相关机制，包含pH-稳态、能量代谢以及转录和蛋白水平的变化。

2.2.1 改变培养条件

LAB在增殖、冷冻干燥、消化过程中受到胁迫之后具有复杂且多样的调控机制^[2]。LAB的培养条件包含温度、pH、碳源、氮源、缓冲盐等，研究人员发现冷冻干燥过程中，与改变冻干参数或使用冷冻保护剂相比，改变培养条件更具有降低成本和提高效率的优势^[75]。Wang等^[76]将嗜酸乳杆菌在30 °C或pH为5.0的条件下培养时，细胞通过改变细胞膜的饱和与不饱和脂肪酸的比例获得了更好的冷冻抗性。在探究碳源成分时，Silva等^[77]发现在保加利亚杆菌的培养基中添加蔗糖后，在增强抗逆性方面比其他碳源更具优势。在Li等^[78]研究中，保加利亚乳杆菌在含吐温-20和蔗糖的培养基中增殖时，由于环丙烷脂肪酸含量的显著增加，冷冻干燥后存活率明显提高。除碳源因素外，氮源对LAB的细胞活性的影响同样受到关注。Shao等^[79]发现，培养基中若含有酵母提取物反而会降低保加利亚乳杆菌的低温耐受性，探究得出酵母提取物会使菌体生长得更长，加大了低温对细胞的损伤，因此冻干存活率降低。同样地，在包维臣^[80]的培养基优化实验中，将氮源酵母浸膏替换成动物蛋白后，菌株存活率更高。

酸胁迫是工业生产和胃肠道消化中遇到的重要生存挑战之一。Broadbent等^[81]在培养基中添加30 mmol/L组氨酸后，干酪乳杆菌通过在胞内积累苹果酸和组氨酸使其在酸性条件下的存活率提高了100倍。Wu等^[82]也发现低pH环境会导致干酪乳杆菌中天冬氨酸和精氨酸的积累，后通过外源添加这2种氨基酸的方式使菌株在pH 3.3条件下的存活率分别提高了1.36倍和2.10倍。Wang等^[83]则通过代谢组学发现pH-稳态机制相关的氨基酸还有赖氨酸、谷氨酸，它们与菌株的黏附能力密切相关。除外源添加氨基酸之外，培养基的酸碱性也是影响LAB抗逆性的一项因素。E等^[84]在探究培养基中的l-谷氨酸冷冻干燥保护机制时，发现酸性环境不仅会上调与细胞膜完整性相关的dnaK基因和secG基因的表达，还会上调murL、murD和vanY基因的表达来增加肽聚糖的合成。E等^[85]还通过调整植物乳杆菌LIP-1培养基的缓冲体系，从转录水平分析出缓冲盐中的K^＋上调了LuxS/autoinducer-2 (AI-2)群体感应系统中luxS基因的表达，促进了生物膜形成，从而赋予目标菌株更高的冻干存活率和更好的储藏稳定性。

除了上述常见的培养基成分外，还可以根据菌株的特异性添加其他能增强其抗逆性的成分。然而，培养条件的调整应该以实际生产为靶向，与生产工艺程序设计相结合才更具有现实意义。

2.2.2 预适应

LAB本身适应压力的能力在抵抗恶劣环境时至关重要。除上述保护策略之外，预适应是将LAB暴露于具有挑战性的环境之前在亚致死条件下适应性培养，已被证明可以有效提高其存活率^[86]。近年来，报道了多项关于通过温和环境的预先适应来提高LAB在各种胁迫条件下的耐受性和活性的工作，在极端压力下，很难通过调节自身的生理代谢来阻止环境因素对菌体的伤害，例如冻干时的低温、人体的消化道环境。表1总结了已报道的不同预适应方式提高菌株抗逆性的机制。

2.2.3 基因工程

近年来，通过转录组学或蛋白质组学方法鉴定了许多与应激保护相关的基因/蛋白质。因此，胁迫耐受性的改善也可以通过基因修饰、诱导已有基因的表达或插入相关基因来实现。对关键基因/蛋白质的表达进行基因工程改造，使LAB在各种胁迫条件下获得更好的活力是一种潜在的策略^[102]。Zhao等^[103]发现重组菌株在酸-乙醇胁迫下，ctsR基因的异源表达使ROS积累减少并增强细胞膜完整性，并提高了Ca²⁺/Mg²⁺-ATP酶和Na⁺/K⁺-ATP酶的活性，首次证明ctsR基因增强了植物乳杆菌的耐酸乙醇性。Darsonval等^[104]则发现，ctsR基因可以充当温度传感器，使得菌株在33 °C以上的生长温度下失活；若抑制酒球菌属中的ctsR基因表达会导致菌株的可培养性显著下降，突出了ctsR在酒球菌属应激反应中的关键作用。Watthanasakphuban等研究利用pSIP表达系统成功构建了2株能够表达活性氧清除酶的重组菌株，能最大限度地减少细胞中ROS的形成，使工业菌株在氧化应激条件下仍能获得较高的细胞密度^[105]。这一发现有益于易受氧化应激的LAB在工厂高密度发酵中的应用。

从基因层面上挖掘菌株耐受性机制之后可以通过对基因进行修饰、诱导、插入来获得具有抗逆性较好的工程菌株，但同时也要面临和解决基因工程的LAB产品所带来的安全隐患。

3 总结与展望

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在LAB的生产、储藏、消化过程中，环境胁迫是不可避免的。为了充分利用LAB在食品工业中作为发酵剂、在医学中作为益生菌、免疫调节剂和活疫苗的潜力，深入了解微生物的复杂防御机制，并根据产品需求设计高活性保护策略，是实现将LAB转化为工业上生产可行性产品的基础。本文总结了LAB从生产到人体摄入一系列过程所遭受的渗透压、温度、氧、酸、胆盐胁迫等的应激反应，并从外源保护和提高菌株本身的抗逆性两大方面阐述了现有的应激保护策略及其机制。调节机制决定了LAB细胞对应激源的敏感性，长期暴露于亚致死剂量的应激源已被证明会在种群水平上引起适应性和微进化的改变。如何根据菌种对不同的应激源进行保护策略的调整对之后LAB产品菌种的筛选和改良，以及生产工艺优化都具有重要指导意义。

目前，许多LAB菌株抗逆性研究的重点聚焦于如何提高环境胁迫下菌株的活力，而菌株的功能特性在经过保护策略处理之后是否改变常常被忽视，LAB功能活性的探究是开发益于人体健康产品的关键。鉴于日渐多元的LAB应用形式，未来的研究可以着重于LAB菌株的保护策略与不同应用产品(如食品)结合后是否仍能发挥原始的功能活性。另外，一项具有实际意义的LAB菌株保护策略，应当是既能与工业生产程序设计紧密结合，又能兼顾到工业上成本的经济性，这样才能实现科学成果向实际生产应用的良好转化。

基金

收起

国家重点研发计划(2023YFF1000302)
广东省自然科学基金(2024A1515010621)
广东省科学院项目(2022GDASZH-2022010101)

参考文献

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文献

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2025年第65卷第2期

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doi: 10.13343/j.cnki.wsxb.20240596

接收时间：2024-09-27
首发时间：2026-02-05
出版时间：2025-02-04

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收稿日期：2024-09-27
录用日期：2024-11-10

基金

National Key Research and Development Program of China(2023YFF1000302)

国家重点研发计划(2023YFF1000302)

Natural Science Foundation of Guangdong Province(2024A1515010621)

广东省自然科学基金(2024A1515010621)

Project of Guangdong Academy of Sciences(2022GDASZH-2022010101)

广东省科学院项目(2022GDASZH-2022010101)

作者信息

1 华南农业大学食品学院，广东广州

3 广东科环生物科技有限公司，广东广州

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Methods	Strains	Term	Results	Mechanisms	References
Heat	Enterococcus faecium	52 ℃ 15 min	Increased tolerance to H₂O₂, ethanol, acid, and alkaline stresses		[4]
	Tetragenococcus halophilus	45 ℃ 1.5 h	Seven-fold increase in survival in ethanol-stressed environments	Involvement in energy metabolism and upregulation of membrane transporter protein genes	[87]
	Lactobacillus acidophilus	65 ℃ 40 min	Increased stability when stored at 37 ℃ and 42 ℃ for one week		[15]
	Lactobacillus acidophilus	45 ℃ 30 min	Freeze-drying survival rate increased from 39.1% to 56.3%	Changes in key enzymes of glycolysis and Na⁺-K⁺ ATPase and increased galactose production	[88]
Cold	Streptococcus thermophilus	10 ℃ 2 h	Increased tolerance to gastric fluids		[89]
	Lactobacillus delbrueckii ssp. bulgaricus	10 ℃ 2 h	Lyophilisation survival rate increased by 16.06%	Expression of two cold shock-induced genes and six heat shock-induced genes were upregulated	[79]
	Lactobacillus kefiranofaciens	20 ℃ 1 h	Inducing cross-stress and increasing resistance to other adversarial environments	Up-regulation of the expression of the molecular chaperones DnaK and GroEl	[90]
	Lactobacillus helveticus	10 ℃ 2 h		Increased synthesis of cyclopropane fatty acids and enhanced cell membrane fluidity	[91]
	Lactobacillus brevis	-5 ℃ 2 h	Extended shelf life	Production of surface proteins	[92]
Acid	Lactobacillus casei	pH 4.510 min	100-fold increase in survival at pH 2.5	Intracellular malate and histidine accumulation	[81]
	Lactobacillus plantarum	pH 3.0 1 h	Increased survival rate after 180 days of storage at room temperature		[93]
	Lactobacillus delbrueckii ssp. bulgaricus	pH 5.0 MRS	Freeze-drying survival rate increased to 68.3%		[79]
	Lactobacillus plantarum	pH 4.5 2 h	Increased lyophilisation survival	Genes involved in fatty acid synthesis and amino acid metabolism and sugar metabolism were significantly upregulated	[94]
NaCl	Lactobacillus acidophilus	0.6 mol/L NaCl		Increased synthesis of surface proteins	[95]
	Lactobacillus plantarum	0.4 mol/L NaCl	Increased lyophilisation survival	Up-regulation of K⁺ transporter-related trkA gene increases cell membrane unsaturated fatty acid content	[96]
	Lactobacillus delbrueckii ssp. bulgaricus	0.2 mol/L NaCl		Increased accumulation of the compatible solute glycine betaine	[97]
	Lactobacillus delbrueckii ssp.bulgaricus	2% NaCl 2 h	Increased glucose utilisation	Increased activity of glycolytic enzymes	[98]
H₂O₂	Bifidobacterium animalis	1.5 mmol/L H₂O₂		Up-regulation of genes encoding thioredoxin systems and divalent cation transporter protein genes	[99]
	Lactobacillus rhamnosus	0.5 mmol/L H₂O₂	Increased survival in sublethal conditions	Enhanced regulation of amino acid metabolism and group sensing pathways	[100]
	Bifidobacterium	210 ppm dissolved oxygen	No loss of viability after 35 days of storage		[101]

Methods

Strains

Term

Results

Mechanisms

References

Heat

Enterococcus faecium

52 ℃ 15 min

Increased tolerance to H₂O₂, ethanol, acid, and alkaline stresses

[4]

Tetragenococcus halophilus

45 ℃ 1.5 h

Seven-fold increase in survival in ethanol-stressed environments

Involvement in energy metabolism and upregulation of membrane transporter protein genes

[87]

Lactobacillus acidophilus

65 ℃ 40 min

Increased stability when stored at 37 ℃ and 42 ℃ for one week

[15]

Lactobacillus acidophilus

45 ℃ 30 min

Freeze-drying survival rate increased from 39.1% to 56.3%

Changes in key enzymes of glycolysis and Na⁺-K⁺ ATPase and increased galactose production

[88]

Cold

Streptococcus thermophilus

10 ℃ 2 h

Increased tolerance to gastric fluids

[89]

Lactobacillus delbrueckii ssp. bulgaricus

10 ℃ 2 h

Lyophilisation survival rate increased by 16.06%

Expression of two cold shock-induced genes and six heat shock-induced genes were upregulated

[79]

Lactobacillus kefiranofaciens

20 ℃ 1 h

Inducing cross-stress and increasing resistance to other adversarial environments

Up-regulation of the expression of the molecular chaperones DnaK and GroEl

[90]

Lactobacillus helveticus

10 ℃ 2 h

Increased synthesis of cyclopropane fatty acids and enhanced cell membrane fluidity

[91]

Lactobacillus brevis

-5 ℃ 2 h

Extended shelf life

Production of surface proteins

[92]

Acid

Lactobacillus casei

pH 4.510 min

100-fold increase in survival at pH 2.5

Intracellular malate and histidine accumulation

[81]

Lactobacillus plantarum

pH 3.0 1 h

Increased survival rate after 180 days of storage at room temperature

[93]

Lactobacillus delbrueckii ssp. bulgaricus

pH 5.0 MRS

Freeze-drying survival rate increased to 68.3%

[79]

Lactobacillus plantarum

pH 4.5 2 h

Increased lyophilisation survival

Genes involved in fatty acid synthesis and amino acid metabolism and sugar metabolism were significantly upregulated

[94]

NaCl

Lactobacillus acidophilus

0.6 mol/L NaCl

Increased synthesis of surface proteins

[95]

Lactobacillus plantarum

0.4 mol/L NaCl

Increased lyophilisation survival

Up-regulation of K⁺ transporter-related trkA gene increases cell membrane unsaturated fatty acid content

[96]

Lactobacillus delbrueckii ssp. bulgaricus

0.2 mol/L NaCl

Increased accumulation of the compatible solute glycine betaine

[97]

Lactobacillus delbrueckii ssp.bulgaricus

2% NaCl 2 h

Increased glucose utilisation

Increased activity of glycolytic enzymes

[98]

H₂O₂

Bifidobacterium animalis

1.5 mmol/L H₂O₂

Up-regulation of genes encoding thioredoxin systems and divalent cation transporter protein genes

[99]

Lactobacillus rhamnosus

0.5 mmol/L H₂O₂

Increased survival in sublethal conditions

Enhanced regulation of amino acid metabolism and group sensing pathways

[100]

Bifidobacterium

210 ppm dissolved oxygen

No loss of viability after 35 days of storage

[101]