简介:动物亚种。乳酸HN019是一种市售的特征良好的益生菌,对人类健康有记载的影响,例如增强免疫功能和平衡肠道微生物组的能力。因此,优化制造过程以提高可持续性,提高生物量产量和生存能力,避免在培养基中使用动物源性营养素,以满足纯素消费者的需求,目前感兴趣。除了确定使用活的益生菌细胞,替代补充剂表示为postbiotics,例如非活细胞和/或益生菌衍生的生物活性分子可能被认为是潜在的下一代生物治疗剂。事实上,postbiotics的优势包括更少的技术限制,例如更简单的生产工艺和扩大规模,甚至更高的特异性。方法:在这项工作中,培养基设计与不同的发酵策略,如分批,在实验室规模的生物反应器上结合了分批补料和原位产物去除。通过超滤和蛋白酶消化进行培养基预处理以减少多糖污染物并促进分泌的胞外多糖(EPS)的纯化。后者从发酵液中分离,并通过NMR表征,GC-MS和SEC-TDA分析。EPS处理的LPS攻击分化CaCo-2细胞中TLR-4、NF-kb和IL-6的表达,活的和热杀死的乳酸双歧杆菌细胞/肉汤,通过蛋白质印迹和ELISA进行体外评估。还通过免疫荧光测定法评估了Zonulin。结果和讨论:通过应用ISPR发酵策略,活的乳酸双歧杆菌HN019的滴度在无动物的半限定培养基上增加到2.9±0.1x1010。中等预处理和简单的下游程序丰富了回收的EPS的代表性(87%),其组成揭示了在由双歧杆菌产生的多糖中通常存在的其他糖中存在甘露糖醛酸。孤立的EPS,首次比较了活细胞和全热灭活肉汤的免疫调节和抗炎特性以及促进肠屏障完整性的能力。有趣的是,EPS和活细胞样品通过下调TLR-4和NF-kb的表达表现出免疫刺激特性,以及通过上调zonulin的表达来促进恢复肠屏障完整性的能力,形成蛋白质的紧密连接之一。以热量杀死肉汤形式的益生菌仅降低NF-kb的表达,而它们在其他测试条件下似乎无效。
Introduction: B. animalis subsp. lactis HN019 is a commercially available well-characterized probiotic with documented effects on human health, such as the ability to enhance the immune function and to balance the intestinal microbiome. Therefore, optimizing the manufacturing process to improve sustainability, increasing biomass yields and viability, and avoiding animal -derived nutrients in the medium to meet vegan consumer\'s needs, is currently of interest. Besides the established use of live probiotic cells, alternative supplements indicated as postbiotics, like non-viable cells and/or probiotics derived bioactive molecules might be considered as potential next generation biotherapeutics. In fact, advantages of postbiotics include fewer technological limitations, such as easier production processes and scale-up, and even higher specificity. Methods: In this work, medium design together with different fermentation strategies such as batch, fed-batch and in situ product removal on lab-scale bioreactors were combined. Medium pretreatment by ultrafiltration and protease digestion was performed to reduce polysaccharidic contaminants and facilitate the purification of secreted exopolysaccharides (EPS). The latter were isolated from the fermentation broth and characterized through NMR, GC-MS and SEC-TDA analyses. The expression of TLR-4, NF-kb and IL-6 in LPS challenged differentiated CaCo-2 cells treated with EPS, live and heat-killed B. lactis cells/broth, was evaluated in vitro by western blotting and ELISA. Zonulin was also assessed by immunofluorescence assays. Results and Discussion: The titer of viable B. lactis HN019 was increased up to 2.9 ± 0.1 x 1010 on an animal-free semidefined medium by applying an ISPR fermentation strategy. Medium pre-treatment and a simple downstream procedure enriched the representativity of the EPS recovered (87%), the composition of which revealed the presence of mannuronic acid among other sugars typically present in polysaccharides produced by bifidobacteria. The isolated EPS, live cells and whole heat inactivated broth were compared for the first up to date for their immunomodulatory and anti-inflammatory properties and for their ability to promote intestinal barrier integrity. Interestingly, EPS and live cells samples demonstrated immune-stimulating properties by downregulating the expression of TLR-4 and NF-kb, and the ability to promote restoring the integrity of the intestinal barrier by up-regulating the expression of zonulin, one of the tight junctions forming proteins. Postbiotics in the form of heat killed broth only reduced NF-kb expression, whereas they did not seem effective in the other tested conditions.