生物膜是附着在生物或非生物表面并生活在自身所产生的胞外聚合物中的复杂微生物群落。很多研究表明，与游离态细菌相比，生物膜态细菌对抗生素、有毒物质及其他不良环境具有更好的适应能力，然而，关于生物膜的大多数研究集中在病原菌的感染过程，而生物膜的有利作用经常被忽视。实验室前期筛选出具有良好成膜能力且对生物胺有显著抑制作用的植物乳杆菌JB1，因此本研究以植物乳杆菌JB1为研究对象，通过优化其生物膜形成条件、制备适宜生物膜生长的载体、探究生物膜菌体在真空冷冻干燥下的保护机制，为益生菌生物膜产业化应用提供指导。具体研究内容及结果如下：

1．为了提高植物乳杆菌JB1生物膜形成，利用结晶紫染色法测定植物乳杆菌JB1生物膜的形成量，并通过单因素实验和正交实验对该菌在不同条件（包括温度、pH、碳源、氮源、金属离子和磷酸氢二钾浓度）下的生物膜形成、产酸性、生长性能等指标进行测定，从而选出最适条件。结果显示：植物乳杆菌JB1具有良好的生物膜形成能力，且该菌的最佳生物膜形成条件为：麦芽糖浓度35 g/L，胰蛋白胨浓5 g/L，锰离子浓度0.2 g/L，磷酸氢二钾浓度10 g/L，培养温度37 ℃，培养液pH6.5。

2．为了制备一种适宜植物乳杆菌JB1形成生物膜的食品级载体，选取魔芋粉为主要材料，利用碱加热能使魔芋葡聚多糖（KGM）形成不可逆凝胶的原理，并结合酵母发酵产生气体，成功制备出发酵多孔魔芋凝胶，并对制备的凝胶载体进行性能表征，测定了载体的溶胀率、质构特性、孔隙率、傅里叶红外光谱和表观粘度。结果显示：随着KGM浓度增加，载体的溶胀率和孔隙率逐渐下降，表观粘度、硬度、回复性和咀嚼性逐渐增加，弹性和内聚性先增加后降低；随着发酵时间的延长，载体的溶胀率和孔隙率逐渐增加，表观粘度、硬度、内聚性、回弹性和咀嚼性逐渐下降，弹性先增加后降低。载体的这种性质和内部结构及交联密度等有很大关系。通过傅里叶红外光谱图可知该材料能够形成不可逆凝胶是由于魔芋葡聚多糖中乙酰基的氧化作用，对不同KGM浓度和发酵时间形成的载体的挂膜性能进行测定，发现当魔芋粉浓度为7%和发酵时间为2 h时载体上黏附的生物膜最多，这也与载体的物理性质具有一定的相关性。将该载体应用于形成生物膜，并在不同环境压力（包括温度、胆盐浓度、人工胃肠液）下比较与游离态细菌的存活优势，发现形成了生物膜的细菌具有更好的环境压力耐受性。

3．为了进一步揭示生物膜在真空冷冻干燥条件下对菌体的保护机制，第三章在第二章的基础上，以植物乳杆菌JB1为研究对象，测定了生物膜态乳酸菌和游离态乳酸菌在冻干不同阶段的各种性质，通过酶活性测定观察细胞活性变化、通过在亚致死浓度溶菌酶和氯化钠条件下测定菌活观察细菌通透性变化、通过傅里叶红外扫描观察细胞成分变化、通过扫描电镜和荧光染色实验观察细胞结构变化、通过荧光定量PCR观察关键基因表达量情况。结果显示：在冻干阶段，生物膜态乳酸菌具有更高的乳酸脱氢酶和β-半乳糖苷酶活力，这两种酶是参与糖酵解及乳糖代谢的关键酶，通过溶菌酶敏感性实验观察菌体细胞壁变化，发现在具有亚致死浓度的溶菌酶存在的情况下，生物膜态乳酸菌具有更高的存活率，说明生物膜态乳酸菌的细胞壁损伤程度较小，氯化钠敏感性实验也说明了生物膜态乳酸菌细胞膜受损较小，从而减少了钠离子和氯离子进入菌体内部，活死细菌染色实验同样也证明上述结果，通过傅里叶红外光谱图研究细胞成分改变情况，发现生物膜态乳酸菌的核酸、蛋白质和碳水化合物成分改变较小。此外，还通过关键基因表达量进一步探讨了冻干过程影响菌体活性的内在机制，结果说明生物膜态乳酸菌提高了galT、PfkA、ldh-1、pdhA、csp、gtaB、sdaC和luxS基因的表达量，这些基因都是参与细胞代谢及群体感应的相关基因，因此，本章实验说明，生物膜菌通过基因调控，改善菌体代谢能力，提高群体感应行为表达，进而调控表观变化，使菌体保持在较好的酶活性、较完整的细胞结构，从而更加适应真空冷冻干燥下极端环境的生存。

4．为了比较生物膜态和游离态乳酸菌在香肠中的发酵效果。将植物乳杆菌JB1接种于发酵香肠中，对发酵香肠的相关感官指标、微生物指标和生物胺含量等指标进行测定。结果显示：添加植物乳杆菌JB1能快速降低香肠pH值，最低能够降到4.81左右，有效抑制了肠杆菌数量，并且加强了蛋白质的降解、小肽与氨基酸态氮的产生，改善了产品的色差值。与游离组相比，生物膜态菌体具有更高的代谢活力，对香肠的生物胺总量也有显著抑制作用，发酵4 d后最高能降低38.89 mg/kg，通过微生物多样性测定，接种发酵剂后，香肠中的细菌OUT数减少，有害菌数量减少，对发酵香肠的安全性起到更积极的效果。

本研究解析了生物膜态菌株抵御冷冻干燥环境的保护机制，制备出适宜菌体成膜的载体，为生物膜在食品中的应用提供了新的研究思路和参考依据。

Biofilm is a complex microbial community that adheres to biological or non biological surfaces and lives in extracellular polymers produced by itself. Many studies have shown that compared to free form bacteria, biofilm form bacteria have better adaptability to antibiotics, toxic substances, and other adverse environments. Therefore, biofilm is a major challenge for researchers in the field of pathogenic bacteria. But its significant characteristics give us inspiration: if we consider from the perspective of probiotics, we can utilize the advantage of biofilms to improve the problem of probiotics being easily damaged by environmental pressure during production and application, thereby greatly improving the application effect of probiotics in production and life. Therefore, this study takes Lactobacillus plantarum JB1 as the research object, and provides guidance for the industrial application of probiotic biofilms by optimizing its biofilm formation conditions, preparing suitable carriers for biofilm growth, and exploring the protective mechanism of biofilm cells under vacuum freeze-drying. The specific research content and results are as follows:

Using crystal violet staining method to determine the formation amount of biofilm of Lactobacillus plantarum JB1, and through single factor and orthogonal experiments, the biofilm formation, acid production, and growth performance of the bacterium under different conditions (including temperature, pH, carbon source, nitrogen source, metal ions, and potassium dihydrogen phosphate concentration) were measured. The optimal conditions were selected. The results showed that Lactobacillus plantarum JB1 has good biofilm formation ability, and the optimal biofilm formation conditions for the bacterium are: maltose concentration of 35 g/L, tryptone concentration of 5 g/L, manganese ion concentration of 0.2 g/L, potassium dihydrogen phosphate concentration of 10 g/L, culture temperature of 37 ℃, and culture medium pH of 6.5.

A kind of gel carrier material suitable for the formation of biofilm by plant lactic acid bacteria JB1 was prepared. The main material of the carrier was konjak flour. The formation of gel was based on the water absorption of konjak glucomannan (KGM) in konjak flour and the characteristics of irreversible gel formation, combined with the principle of gas production by yeast fermentation, to form a green and stable porous gel. The performance of the prepared gel carrier was characterized, and the swelling rate, texture characteristics, porosity, Fourier infrared spectrum and apparent viscosity of the carrier were measured. The results showed that as the concentration of KGM increased, the swelling rate and porosity of the carrier gradually decreased, while the apparent viscosity, hardness, recovery, and chewiness gradually increased. The elasticity and cohesion first increased and then decreased; As the fermentation time prolongs, the swelling rate and porosity of the carrier gradually increase, while the apparent viscosity, hardness, cohesion, resilience, and chewiness gradually decrease, and the elasticity first increases and then decreases. The properties of the carrier are closely related to its internal structure and crosslinking density. It can be seen from the Fourier infrared spectrum that the material can form irreversible gel due to the oxidation of acetyl group in konjac glucomannan. The film hanging performance of the carrier formed by different KGM concentration and fermentation time was measured. It was found that when the konjac powder concentration was 7% and the fermentation time was 2 h, the biofilm adhered to the carrier was the most, which was also related to the physical properties of the carrier. The carrier was applied to form biofilms, and the survival advantages of free bacteria were compared under different environmental pressures (including temperature, bile salt concentration, and artificial gastrointestinal fluid). It was found that bacteria that formed biofilms had better environmental pressure tolerance.

In order to further reveal the protective mechanism of biofilm on bacterial cells under vacuum freeze-drying conditions, Chapter 3, based on Chapter 2, measured the various properties of biofilm lactic acid bacteria and free lactic acid bacteria at different stages of freeze-drying, including enzyme activity, changes in cell wall and membrane, and changes in cell composition. The results showed that biofilm lactic acid bacteria had higher activity of lactate dehydrogenase and β - galactosidase during the freeze - drying stage, which were key enzymes involved in glycolysis and lactose metabolism. Through lysozyme sensitivity experiments, changes in bacterial cell wall were observed, and it was found that lysozyme with sublethal concentration had higher activity. In this case, biofilm lactic acid bacteria have a higher survival rate, indicating that the degree of cell wall damage in biofilm lactic acid bacteria is relatively small, the sodium chloride sensitivity experiment also showed that the cell membrane of biofilm lactic acid bacteria was less damaged, thereby reducing the entry of sodium and chloride ions into the interior of the bacterial body. The staining experiment of live and dead bacteria also confirmed the above results. By studying the changes in cell composition through Fourier transform infrared spectroscopy, it was found that the changes in nucleic acid, protein, and carbohydrate components of biofilm lactic acid bacteria were relatively small. In addition, the intrinsic mechanism by which the freeze-drying process affects bacterial activity was further explored through the expression levels of key genes. The results showed that biofilm lactic acid bacteria increased the expression levels of galT, PfkA, ldh-1, pdhA, csp, gtaB, sdaC, and luxS genes, all of which are related genes involved in cell metabolism and quorum sensing. Therefore, this chapter's experiment demonstrates that biofilm bacteria improve their metabolic ability and quorum sensing behavior expression through gene regulation, thereby regulating epigenetic changes and maintaining good enzyme activity and complete cell structure, thus better adapting to the survival of extreme environments under vacuum freeze-drying.

Applying Lactobacillus plantarum JB1 as a fermenting agent to fermented sausages, comparing the inhibitory effects of biofilm and free states on the properties of fermented sausages and biogenic amines. The results showed that adding Lactobacillus plantarum JB1 could quickly reduce the pH value of sausages, with a minimum of around 4.81, effectively inhibiting the number of Enterobacteriaceae and enhancing protein degradation, the production of small peptides and amino acid nitrogen, and improving the color difference value of the product. Compared with the free group, biofilm form bacteria have higher metabolic activity and a significant inhibitory effect on the total amount of biogenic amines in sausages. After four days of fermentation, the maximum decrease can be 38.89mg/kg. Through microbial diversity testing, after inoculation with fermentation agents, the bacterial OUT number in sausages decreases and the number of harmful bacteria decreases, which has a more positive effect on the safety of fermented sausages.

This study analyzed the protective mechanism of biofilm strains against freeze-drying environment, prepared suitable carriers for bacterial film-forming, and provided new research ideas and reference basis for the application of biofilm in food.

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