Name: 益生菌喷雾干燥的工艺开发和产品质量评估 (视频) | JoVE Journal
Uploaded: 2023-04-07T14:45:00.000+00:00
Duration: 5 min 45 s
Description: 了解喷雾干燥微胶囊化技术用于益生菌生产及其理化特性表征的方法。本研究展示了如何优化工艺条件以提高益生菌存活率，为功能性食品开发提供有效策略。

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5 minutes

本研究部分由巴西高等教育人员提升协调办公室（Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil，CAPES）资助，资助编号为001。本研究还部分得到了圣保罗研究基金会（FAPESP）的支持。E.C.P.D.M. 感谢国家科学技术发展委员会（CNPq）提供的研究员奖学金 306330/2019-9。

1. 益生菌细胞的制备
<ol>
	<li>制备脱曼罗加斯沙普（MRS）肉汤。</li>
	<li>在MRS肉汤中复苏目标菌种1%（v/v）的培养物（本实验中使用的是植物乳杆菌Lactiplantibacillus paraplantarum FT-259）。</li>
	<li>在适宜温度下培养24小时（我们采用37 °C）。</li>
</ol>
2. 从培养物中分离细菌
<ol>
	<li>使用50 mL锥形管，在4 °C条件下以7,197 × g离心5分钟，收集菌体。离心前必须确保管体重量平衡。</li>
	<li>用移液器吸除上清液，并将其弃置在合适的容器中。用磷酸盐缓冲液（pH 7）洗涤沉淀，并将溶液充分混匀。</li>
	<li>重复前述离心步骤。</li>
	<li>为获得沉淀，使用移液器将上清液吸出并弃置于适当容器中。</li>
</ol>
3. 添加干燥助剂
<ol>
	<li>选择两种干燥助剂（保护剂）组合：菊粉：麦芽糊精混合物和模拟脱脂乳（表1）22,23。</li>
	<li>称取5 g菊粉和5 g麦芽糊精，得到第一种保护剂组合。</li>
	<li>称取3 g菊粉、3 g乳糖、0.4 g胶体二氧化硅（SiO2）和3.6 g乳清蛋白，得到第二种保护剂组合。</li>
	<li>将每种干燥助剂加入超纯水（1:10）中，并进行磁力搅拌直至完全溶解。</li>
	<li>确保保护剂与水形成均匀溶液后，将益生菌菌体颗粒加入该混合液中，温和搅拌20分钟。</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>干燥助剂</td>
			<td>菊粉和麦芽糊精</td>
			<td>模拟脱脂乳</td>
		</tr>
		<tr>
			<td>麦芽糊精</td>
			<td>5%</td>
			<td>-</td>
		</tr>
		<tr>
			<td>乳清蛋白</td>
			<td>-</td>
			<td>3.60%</td>
		</tr>
		<tr>
			<td>乳糖</td>
			<td>-</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>菊粉</td>
			<td>5%</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>胶体 SiO2</td>
			<td>-</td>
			<td>0.40%</td>
		</tr>
	</tbody>
</table>
表1：干燥助剂的组成。
4. 喷雾干燥
<ol>
	<li>开启喷雾干燥机（SD），并按以下参数设置干燥气体流速、进气温度、雾化气体流速和压力： 
	进气温度：80 °C 
	空气流量：60 m³/h 
	进料速率：4 g/min 
	雾化流量：17 L/min 
	雾化压力：1.5 kgf/cm² 
	雾化喷嘴直径：1 mm</li>
	<li>配制保护剂成分，并加入浓缩的益生菌菌体沉淀。</li>
	<li>通过蠕动泵开始进料益生菌混合物（细胞加保护剂）。</li>
	<li>当溶液进入雾化器时，启动计时器，并放置产品收集容器。</li>
	<li>每5分钟记录一次出口气体温度，以监测可能的温度波动。</li>
	<li>当全部益生菌混合物完成进料后，停止计时。</li>
	<li>称量产品收集容器，以确定进入系统的物料量和收集到的干燥产物量，通过干燥机内的物料平衡计算干燥得率（回收产物量）。</li>
	<li>使用模拟脱脂乳评估温度对益生菌细胞活性的影响，设置五种不同的喷雾干燥温度（80 °C、100 °C、120 °C、140 °C 和 160 °C，对应的出口气温分别为&#160;59 °C、70 °C、83 °C、96 °C 和 108 °C）。</li>
</ol>
5. 粉末表征
<ol>
	<li>产品水分含量
	<ol>
		<li>精确称取100 mg干燥产品，置于卡尔·费休水分测定仪的滴定池中。</li>
		<li>按下启动按钮，开始对样品中的水分进行双安培滴定。</li>
	</ol>
	</li>
	<li>水分活度
	<ol>
		<li>在25 °C下，称取0.6 g干燥产品放入湿度计的样品室中。</li>
		<li>关闭仪器盖子。 
		&#8203;注意：当样品在样品室内达到平衡蒸气压时，测试将自动开始并停止。</li>
	</ol>
	</li>
</ol>
6. 益生菌活性
<ol>
	<li>将先前制备的细菌悬液用9 mL蛋白胨水（0.1%，v/v）进行稀释。</li>
	<li>涡旋振荡至完全分散。</li>
	<li>在9 mL生理盐水（0.9% NaCl）中进行连续十倍系列稀释（1:10）。</li>
	<li>将各稀释液接种至MRS琼脂平板，于37 °C培养24–48 h。</li>
	<li>使用带放大镜的菌落计数器计数菌落形成单位（CFU/g）。</li>
	<li>根据以下公式计算干燥产品中益生菌的存活率： 
	EE (%) = (N∕No) × 100 
	其中，N为喷雾干燥后活菌数，No为喷雾干燥前细菌细胞数。</li>
	<li>以每克产品分散液中的CFU数表示活菌数量。</li>
</ol>
7. 数据分析
<ol>
	<li>将获得的数据整理到统计软件中，并使用多重比较检验（ANOVA）进行分析。</li>
</ol>

Optimizing Spray-Dried Probiotic Product Quality Through Process Development

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<li>Labuza, T. P., Altunakar, B. Water activity prediction and moisture sorption isotherms. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLabuza%2C+TP+%22Water+Activity+in+Foods%3A+Fundamentals+and+Applications%22">Water Activity in Foods: Fundamentals and Applications</a>. Barbosa-Canovas, G. V., Fontana, A. J., Schmidt, S. J., Labuza, T. P. , John Wiley & Sons, Inc. Hoboken, NJ. 161-205 (2020).</li>
<li>Misra, S., Pandey, P., Mishra, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+approaches+for+co-encapsulation+of+probiotic+bacteria+with+bioactive+compounds%2C+their+health+benefits+and+functional+food+product+development%3A+A+review%5BTitle%5D)+AND+%22Trends+in+Food+Science+%26+Technology%22%5BJournal%5D)">Novel approaches for co-encapsulation of probiotic bacteria with bioactive compounds, their health benefits and functional food product development: A review.</a> Trends in Food Science & Technology. 109, 340-351 (2021).</li>
<li>Misra, S., Pandey, P., Dalbhagat, C. G., Mishra, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+technologies+and+coating+materials+for+improved+probiotication+in+food+products%3A+A+review%5BTitle%5D)+AND+%22Food+and+BioprocessTechnology%22%5BJournal%5D)">Emerging technologies and coating materials for improved probiotication in food products: A review.</a> Food and BioprocessTechnology. 15 (5), 998-1039 (2022).</li>
<li>Martins, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+ideal+water+activity+and+powder+temperature+after+spray+drying+to+reduce+Lactococcus+lactis+cell+viability+loss%5BTitle%5D)+AND+%22Journal+of+Dairy+Science%22%5BJournal%5D)">Determination of ideal water activity and powder temperature after spray drying to reduce Lactococcus lactis cell viability loss.</a> Journal of Dairy Science. 102 (7), 6013-6022 (2019).</li>
<li>Vock, S., Klöden, B., Kirchner, A., Weißgärber, T., Kieback, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Powders+for+powder+bed+fusion%3A+A+review%5BTitle%5D)+AND+%22Progress+in+Additive+Manufacturing%22%5BJournal%5D)">Powders for powder bed fusion: A review.</a> Progress in Additive Manufacturing. 4, 383-397 (2019).</li>
</ol>

作者声明无利益冲突。

L. paraplantarum FT-259 是一种革兰氏阳性、杆状细菌，可产生具有抗单核细胞增生李斯特菌活性的细菌素，且具有较高的益生潜力20。Son 等人24此前已证实 L. paraplantarum 菌株具有免疫刺激和抗氧化能力。此外，该菌株还具有良好的益生特性，例如在人工胃液和胆汁条件下具有稳定性、对抗生素敏感以及能够与肠道细胞结合。另外，其不产生可能对胃肠道产生负面影响的代谢产物。Choi 和 Chang25研究了L. plantarum EM，并基于其胆盐水解酶活性和细胞表面结合能力，报道了该菌株在降低胆固醇方面的潜力。除了对酸性和胆汁应激具有耐受性外，L. plantarum EM 还表现出对病原菌的抗菌活性以及对抗生素的耐药性，进一步验证了其作为益生菌的应用潜力。
然而，将益生菌制成干燥产品以实现商业化颇具挑战性，因为微生物会暴露于多种应激因素下，例...

要被定义为益生菌，添加到食品（或补充剂）中的微生物必须以活体形式摄入，能够在宿主胃肠道中存活，并以足够的数量到达作用部位以发挥有益作用1,2,7。
人们对益生菌日益增长的兴趣源于其对人类健康的多种益处，例如通过抑制有害微生物来刺激免疫系统、降低血清胆固醇水平、增强肠道屏障功能，以及在治疗肠易激综合征等方面的有益作用2,3。此外，多项研究表明，益生菌还可对体内其他因微生物群失衡而引发感染性疾病的部位产生积极影响3,4,5。
为了使益生菌具有治疗效果，产品在食用时应含有 106-107 CFU/g 的细菌6。另一方面，意大利卫生部和加拿大卫生部已规定，食品中益生菌的最低含量应分别为每天或每份 109 CFU/g 的活菌细胞7。鉴于需要较高剂量的益生菌以确保其产生有益作用，必须保障益生菌在加工过程、货架储存期间以及通过胃肠道（GI）过程中存活。多项研究表明，微胶囊化是一种有效提高益生菌整体存活性的方法8,9,10,11。
在此背景下，已开发出多种益生菌微胶囊化方法，例如喷雾干燥、冷冻干燥、喷雾冷却、乳化、挤压、凝聚法，以及近年来应用的流化床技术11,12,13,14。其中，喷雾干燥（SD）法在食品工业中被广泛应用，因其操作简单、快速且可重复。该方法易于放大，生产效率高，且能耗较低11,12,13,14。然而，高温和低水分环境可能影响益生菌细胞的存活率和活性15。针对特定菌株，可通过研究培养物的培养时间及培养条件，对菌体进行预适应，并优化喷雾干燥参数（进风和出风温度、雾化过程）以及包埋材料的组成，从而改善上述两个因素对菌体的影响8,14,16,17,18。
包埋溶液的组成在喷雾干燥（SD）过程中也是一个重要因素，因为它可以决定对不利环境条件的保护程度。菊粉、阿拉伯胶、麦芽糊精和脱脂乳被广泛用作益生菌干燥的包埋剂5,17,18,19。菊粉是一种具有强效益生元活性的果寡糖，有助于促进肠道健康19。脱脂乳在维持干燥细菌细胞活性方面非常有效，并能产生具有良好复溶性能的粉末17。
Lactiplantibacillus paraplantarum FT-259 是一种产细菌素、具有抗单核细胞增生李斯特菌活性且具备益生特性的乳酸菌20,21。该菌为兼性异型发酵、杆状、革兰氏阳性细菌，可在 15 °C 至 37 °C 范围内生长20，与机体稳态体温相容。本研究旨在系统阐述喷雾干燥益生菌（L. paraplantarum FT-259）的制备及其理化特性表征的全部步骤，并评估保护剂种类与干燥温度对产品性能的影响。

<table><tbody><tr><td>Aqua Lab 4TEV</td><td>Decagon Devices</td><td>-</td><td>水分活度测定仪</td></tr><tr><td>离心机（型号 5430 R）</td><td>Eppendorf</td><td>-</td><td>离心机</td></tr><tr><td>胶体 SiO&lt;sub&gt;2&lt;/sub&gt;（Aerosil 200）</td><td>Evokik</td><td>7631-86-9</td><td>干燥助剂</td></tr><tr><td>菊苣来源的低聚果糖</td><td>Sigma-Aldrich</td><td>9005-80-5</td><td>干燥助剂</td></tr><tr><td>GraphPad Prism（版本 8.0）软件</td><td>GraphPad Software</td><td>-</td><td>美国加利福尼亚州圣地亚哥</td></tr><tr><td>Karl Fischer 870 Titrino Plus</td><td>Metrohm</td><td>-</td><td>水分含量测定</td></tr><tr><td>乳糖</td><td>Milkaut</td><td>63-42-3&amp;nbsp;</td><td>干燥助剂</td></tr><tr><td>麦芽糊精</td><td>Ingredion</td><td>9050-36-6</td><td>干燥助剂</td></tr><tr><td>Milli-Q</td><td>Merk</td><td>-</td><td>超纯水系统</td></tr><tr><td>MRS 琼脂</td><td>Oxoid</td><td>-</td><td>培养基</td></tr><tr><td>MRS 肉汤</td><td>Oxoid</td><td>-</td><td>培养基</td></tr><tr><td>OriginPro（版本 9.0）软件</td><td>OriginLab</td><td>-</td><td>美国马萨诸塞州北安普顿</td></tr><tr><td>喷雾干燥仪 SD-05</td><td>Lab-Plant Ltd</td><td>-</td><td>喷雾干燥仪</td></tr><tr><td>乳清蛋白</td><td>Arla Foods Ingredients S.A.</td><td>91082-88-1</td><td>干燥助剂</td></tr></tbody></table>

益生菌喷雾干燥工艺开发及产品质量评价

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

益生菌和益生元因其对健康的益处而受到食品和制药行业的广泛关注。益生菌是指能够对人类和动物健康产生有益作用的活菌，而益生元则是一类可促进有益肠道菌群生长的营养物质。益生菌粉剂因其服用方便且易于作为膳食补充剂融入日常饮食，近年来广受欢迎。然而，干燥过程会影响菌体的存活率，因为高温会使益生菌失活。在此背景下，本研究旨在系统阐述喷雾干燥益生菌的制备及其理化特性表征的全部步骤，并评估保护剂（模拟脱脂乳及菊粉：麦芽糊精组合）和干燥温度对提高粉末得率及菌体存活率的影响。结果表明，在80 °C条件下，模拟脱脂乳能显著提高益生菌的存活率。使用该保护剂时，随着进风温度升高，益生菌的存活率、水分含量和水活度（Aw）均呈下降趋势。益生菌的存活率与干燥温度呈相反关系。&#160;在接近120 °C的温度下，干燥后的益生菌存活率约为90%，水分含量为4.6% w/w，Aw为0.26，这些数值均处于可确保产品稳定性的适宜范围内。因此，为保障粉末制剂中微生物细胞的存活率、货架期以及在食品加工和储存过程中的存活性，喷雾干燥温度需高于120 °C。

本研究中，使用食品级包埋剂（菊粉：麦芽糊精和模拟乳粉）通过喷雾干燥法对 L. paraplantarum进行包埋，所得产品品质高，并能有效维持细菌细胞的存活率17,19。
益生菌在80 °C下的存活率结果显示，不同的保护体系（菊粉：麦芽糊精和模拟脱脂乳）均能有效保护益生菌细胞，存活率分别为95.1%和97.0%。两种保护体系的产品得率均接近50% w/w，其中模拟脱脂乳略优，所得产品外观和流动性更佳。随后，将益生菌与模拟脱脂乳组合的配方在80 °C至160 °C的更高温度下进行喷雾干燥处理（图1）。
正如预期，SD 温度的升高倾向于降低益生菌的存活率，在 160 °C 时存活率降至近 80%。从图 1中还可以看出，干燥温度对产品得率的影响可忽略不计，平均值为 50.7% ± ...

本方案详细介绍了喷雾干燥益生菌产品的制备及其理化表征步骤。

益生菌和益生元因其对健康的益处而受到食品和制药行业的广泛关注。益生菌是指能够存活并进入肠道后发挥有益作用的活菌……

在我们的实验室中，我们致力于研究有益微生物，包括益生菌。益生菌是一类可添加到食品中的活体微生物，通常通过在肠道内与致病菌竞争以抑制其生长，从而缓解腹泻或其他胃肠道紊乱等症状，进而改善消费者健康。如今，已有多种技术被应用于确保益生菌在食品保质期内的存活。此外，益生菌还需耐受胃肠道中的严苛环境，以在肠道内发挥其有益作用。我们研究了多种可提高益生菌在食品或人体宿主中存活率的技术。在这些技术中，喷雾干燥法微胶囊化因其操作简单、快速、可重复，且能在低能耗下获得高产率而备受关注。然而，确定获得干燥益生菌的合适条件仍具挑战性。为此，必须选择合适的保护剂。脱脂牛奶、麦芽糊精或其他类似益生元的菊粉已用于不同益生菌菌株。微胶囊化领域是一个蓬勃发展的研究方向，有望通过提供可作为功能性成分使用的新产品来影响食品工业。本研究中，我们演示了如何利用喷雾干燥技术制备一种潜在益生菌菌株的微胶囊制剂。同时展示了如何确定表征粉末的主要参数，制备该粉末，以及如何评估益生菌的存活率。

process-development-for-spray-drying-probiotic-bacteria-evaluation

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JoVE Journal

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生物学

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