益生菌喷雾干燥的工艺开发和产品质量评估
Summary
该协议详细介绍了喷雾干燥益生菌产品的生产和物理化学表征所涉及的步骤。
Abstract
益生菌和益生元因其健康益处而受到食品和制药行业的极大兴趣。益生菌是活细菌，可以对人类和动物的健康产生有益的影响，而益生元是喂养有益肠道细菌的营养素。粉末益生菌因其摄入和作为食品补充剂纳入饮食的简便性和实用性而广受欢迎。然而，干燥过程会干扰细胞活力，因为高温会使益生菌失活。在此背景下，本研究旨在介绍喷雾干燥益生菌的生产和物理化学表征所涉及的所有步骤，并评估保护剂（模拟脱脂牛奶和菊粉：麦芽糊精缔合）和干燥温度对提高粉末产量和细胞活力的影响。结果表明，模拟脱脂牛奶在80 °C时提高了益生菌活力。 使用这种保护剂，只要入口温度升高，益生菌活力、水分含量和水分活度 （Aw） 就会降低。益生菌的活力随着干燥温度的升高而降低。在接近120°C的温度下，干燥的益生菌显示出约90%的活力，水分含量为4.6% w/w，Aw为0.26;足以保证产品稳定性的值。在这种情况下，需要高于120°C的喷雾干燥温度，以确保微生物细胞在粉末制剂中的活力和保质期以及食品加工和储存过程中的存活率。
Introduction
要定义为益生菌，添加到食物（或补充剂）中的微生物必须被活活消耗，能够在宿主胃肠道中通过期间存活，并以足够的量到达作用部位以发挥有益作用1，2，7。
对益生菌的兴趣日益浓厚，是因为它们对人类健康具有多种益处，例如刺激免疫系统、降低血清胆固醇水平、通过作用于有害微生物来增强肠道屏障功能，以及它们在治疗肠易激综合征方面的有益作用， 其中2，3。此外，多项研究表明，益生菌可以对人体其他部位产生积极影响，其中不平衡的微生物群落可引起传染病3，4，5。
为了使益生菌具有治疗效果，产品在食用时应含有 10 6-10 7 CFU/g 的细菌6。另一方面，意大利卫生部和加拿大卫生部已确定，食品中益生菌的最低水平应为每天或每份109 CFU / g活细胞，分别为7。考虑到需要大量益生菌来保证它们会产生有益的效果，因此必须保证它们在加工、货架储存和通过胃肠道 （GI） 过程中的存活。多项研究表明，微胶囊化是提高益生菌整体活力的有效方法8，9，10，11。
在这种情况下，已经开发了几种用于益生菌微胶囊化的方法，例如喷雾干燥，冷冻干燥，喷雾冷却，乳液，挤出，凝聚，以及最近的流化床11，12，13，14。喷雾干燥（SD）微胶囊化广泛用于食品工业，因为它是一种简单，快速且可重复的过程。它易于放大，并且在低能量需求下具有高产量11，12，13，14。尽管如此，暴露在高温和低水分含量下会影响益生菌细胞的存活和活力15。对于给定菌株，可以通过确定培养年龄和条件的影响来改善这两个参数，以预先适应培养并优化喷雾干燥条件（入口和出口温度，雾化过程）和封装组合物8，14，16，17，18。
封装溶液的成分也是SD过程中的一个重要因素，因为它可以定义对不利环境条件的保护水平。菊粉、阿拉伯胶、麦芽糊精和脱脂牛奶被广泛用作益生菌干燥的封装剂5，17，18，19。菊粉是一种低聚果糖，具有很强的益生元活性并促进肠道健康19。脱脂牛奶在维持干燥细菌细胞的活力方面非常有效，并产生具有良好复溶性能的粉末17。
植物寄乳杆菌FT-259 是一种乳酸菌，除了益生菌性状20，21 外，还产生细菌素并具有抗李斯特活性。它是一种兼性杂发酵棒状革兰氏阳性细菌，从15°C生长到37°C20，与稳态体温相容。本研究旨在介绍喷雾干燥益生菌（植物乳杆菌FT-259）的生产和物理化学表征所涉及的所有步骤，并评估保护剂和干燥温度的影响。
Protocol
1. 益生菌细胞的生产 准备德曼罗戈萨和夏普（MRS）肉汤。 重新激活MRS肉汤中目标培养物的1%（v / v）（此处使用植物 副植物乳杆菌 FT-259）。 在足够的温度下孵育24小时（我们使用37°C）。 2. 将细菌与培养物分离 使用50mL锥形管在4°C下以7，197× g 离心细菌培养物5分钟。重要的是在手术前平衡管子的重量?…
Representative Results
本研究采用食品级包封剂（菊粉：麦芽糊精和模拟奶粉）对 植物乳杆菌 进行包封，在保持细菌细胞活力方面表现出较高的产品质量和功效17，19。 80 °C下益生菌SD结果显示，不同的保护剂体系（菊粉：麦芽糊精和模拟脱脂乳）促进了益生菌细胞的有效保护，活力分别为95.1%和97.0%。两种保护剂系统的产品收率接近 50% w/w，模拟?…
Discussion
植物拉松 FT-259是一种革兰氏阳性杆状细菌，是具有抗李斯特活性的细菌素的生产者，具有很高的益生菌潜力20。Son等人24 先前证明了 副植物乳杆菌 菌株的免疫刺激剂和抗氧化能力。此外，它们具有巨大的益生菌潜力，具有在人工胃和胆汁条件下的稳定性、对抗生素的敏感性以及与肠道细胞结合等特性。此外，它们不会产生可能对胃肠道产生负面?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究的部分资金来自巴西高级国家财政代码001。这项研究也得到了FAPESP – 圣保罗研究基金会的部分支持。E.C.P.D.M.感谢国家科学技术发展委员会（CNPq）306330/2019-9的研究员奖学金。
Materials
| Aqua Lab 4TEV | Decagon Devices | – | Water activity meter |
| Centrifuge (mod. 5430 R ) | Eppendorf | – | Centrifuge |
| Colloidal SiO2 (Aerosil 200) | Evokik | 7631-86-9 | drying aid |
| Fructooligosaccharides from chicory | Sigma-Aldrich | 9005-80-5 | drying aid |
| GraphPad Prism (version 8.0) software | GraphPad Software | – | San Diego, California, USA |
| Karl Fischer 870 Titrino Plus | Metrohm | – | Moisture content |
| Lactose | Milkaut | 63-42-3 | drying aid |
| Maltodextrin | Ingredion | 9050-36-6 | drying aid |
| Milli-Q | Merk | – | Ultrapure water system |
| MRS Agar | Oxoid | – | Culture medium |
| MRS Broth | Oxoid | – | Culture medium |
| OriginPro (version 9.0) software | OriginLab | – | Northampton, Massachusetts, USA |
| Spray dryer SD-05 | Lab-Plant Ltd | – | Spray dryer |
| Whey protein | Arla Foods Ingredients S.A. | 91082-88-1 | drying aid |
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Cite This Article
Kakuda, L., Jaramillo, Y., Niño-Arias, F. C., Souza, M. F. d., Conceição, E. C., Alves, V. F., Almeida, O. G. d., De Martinis, E. C. P., Oliveira, W. P. Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality. J. Vis. Exp. (194), e65192, doi:10.3791/65192 (2023).