CN112772928B - 一种发酵腐竹豆清液的制备方法及其在促进肠道健康中的应用 - Google Patents
一种发酵腐竹豆清液的制备方法及其在促进肠道健康中的应用 Download PDFInfo
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Abstract
本发明公开了一种发酵腐竹豆清液的制备方法，该方法是将腐竹豆清液经高温灭菌后接种植物乳杆菌菌液，然后在35‑40℃恒温条件下静态发酵2‑5天。本发明还公开了发酵腐竹豆清液在促进肠道健康中的应用以及一种用于促进肠道健康的微胶囊制剂。本发明制备的发酵腐竹豆清液对肠道短链脂肪酸以及有益菌的产生具有促进作用，对有害菌具有抑制作用，从而能促进动物肠道健康，可作为膳食补充剂在食品领域有潜在的利用价值，并且可作为饲料添加剂，在畜牧养殖业中也具有广阔的应用前景。
Description
技术领域
本发明涉及一种发酵腐竹豆清液的制备方法，本发明还涉及该方法制备的发酵腐竹豆清液在促进肠道健康中的应用以及一种用于促进肠道健康的微胶囊制剂。
背景技术
腐竹豆清液是腐竹生产过程中产生的废水。我国的豆制品加工厂大多为中小企业或家庭式的小作坊，生产规模不大，废水大多采用直接排放的方式，造成环境污染和资源浪费。研究表明，腐竹豆清液中含有大量的营养物质，其主要成分是蛋白质、脂肪、糖类等成分，还含有大豆异黄酮、大豆多糖、大豆低聚糖等功能性成分，是一种天然的复合益生元，在调节动物肠道菌群中能发挥一定作用。
目前，对腐竹豆清液进行的研究主要集中在提取功能成分和回收利用等方面，在腐竹豆清液促进肠道功能等保健作用方面的研究处于空白阶段。本发明针对腐竹豆清液及其发酵产物进行功能研究，旨在开发一种能够有效促进动物肠道短链脂肪酸以及调节肠道有益菌丰度，从而具有肠道保健功能的新产品，同时提高资源利用率和减少避免环境污染。
发明内容
本发明的目的是针对上述问题，提供一种发酵腐竹豆清液的制备方法，该方法制备的发酵腐竹豆清液具有更好的促进肠道短链脂肪酸和调节肠道菌群作用，从而不仅实现了腐竹加工副产物的回收利用，减少了资源浪费和环境污染，同时能更好地促进动物肠道健康。
上述目的是通过以下技术方案实现的：
一种发酵腐竹豆清液的制备方法，该方法是：将腐竹豆清液经高温灭菌后接种植物乳杆菌(Lactobacillus plantarum)菌液，然后在35-40℃恒温条件下静态发酵2-5天。
优选地，所述植物乳杆菌菌液的接种量为腐竹豆清液体积的3-10％，菌液中的活菌数为106～109CFU/mL。
进一步优选地，所述植物乳杆菌菌液的接种量为腐竹豆清液体积的6％，菌液中的活菌数为108CFU/mL。
试验表明，发酵腐竹豆清液具有更高的药理活性，产品中所含有的苷元型大豆异黄酮如大豆素、大豆黄素和染料木素的含量均高于发酵前，而且产品在经过体外模拟胃-小肠-结肠消化后，能显著促进肠道短链脂肪酸的产生并能有效调节肠道菌群失调，活性也优于发酵前。以上结果表明本发明制备的发酵腐竹豆清液具有更好的促进动物肠道健康作用。
一种促进肠道健康的微胶囊制剂，其活性成分是按上述方法制备的发酵腐竹豆清液。
一种制备所述微胶囊制剂的方法，该方法包括以下步骤：
(1)将按上述方法制备的发酵腐竹豆清液浓缩后真空冷冻干燥，得冻干粉；
(2)配制浓度为2％的海藻酸钠溶液和壳聚糖浓度为1.5％、氯化钙浓度为3％的壳聚糖-CaCl2混合溶液；
(3)向海藻酸钠溶液中加入6％的发酵腐竹豆清液冻干粉，搅拌混匀；
(4)向壳聚糖-CaCl2混合溶液中滴加NaOH溶液，同时用注射器将步骤(3)得到的溶液匀速滴入壳聚糖-CaCl2混合溶液中，发生交联反应，过滤，干燥，即得包埋发酵腐竹豆清液的微胶囊。
本发明具有以下有益效果：
本发明制备的发酵腐竹豆清液对肠道短链脂肪酸以及有益菌的产生具有促进作用，对有害菌具有抑制作用，从而能促进动物肠道健康，可作为膳食补充剂在食品领域有潜在的利用价值，并且可作为饲料添加剂，在畜牧养殖业中也具有广阔的应用前景。本发明还能够提高腐竹加工过程中的副产物利用率，减少浪费，降低环境污染，具有很好的经济与社会效益前景。
附图说明
图1是测定发酵腐竹豆清液冻干粉含量时绘制的标准曲线。
图2是发酵腐竹豆清液微胶囊在体外模拟胃-小肠消化过程的释放率。
具体实施方式
以下结合具体实施例对本发明进行详细说明，实施例中所涉及的百分比除非另有说明，均指的是重量比。
实施例1腐竹豆清液经发酵后大豆异黄酮的含量变化
腐竹豆清液是腐竹生产过程中获得的加工副产物。将腐竹豆清液经121℃灭菌20分钟，将植物乳杆菌(Lactobacillus plantarum)进行活化和扩大培养，至活菌数为108CFU/mL，将菌液以6％(体积)的接种量接种到上述腐竹豆清液中，在37℃恒温条件下静态发酵3天。
将发酵前和发酵后的腐竹豆清液经80％(体积)甲醇超声提取，离心取上清液，然后采用高效液相色谱法测定大豆异黄酮含量，色谱柱为Agilent Zorbax SB-C18柱(250×4.6mm，5μm)，流动相A为乙腈，流动相B为0.1％甲酸/水(v/v)，流速为1.0mL/min，样品进样量为20μL，检测波长为260nm。梯度洗脱方法：0-5min，15％A；5-28min，35％A；28-42min，45％A；42-47min，90％A；47-59min，15％A。
从表1可知，经过植物乳杆菌发酵后的腐竹豆清液中，苷元型的大豆异黄酮如大豆素、大豆黄素和染料木素的含量显著增加，而糖苷型的大豆异黄酮如大豆苷、大豆黄苷和染料木苷含量显著减少，说明发酵促进了结合型糖苷的水解，从而使苷元型的大豆异黄酮含量增加，而已有文献表明苷元型的大豆异黄酮具有更好的药理活性。
表1发酵处理对腐竹豆清液大豆异黄酮含量的影响(μmol/L)
注：(1)同一列不同角标字母的数据具有显著差异(P＜0.05)
(2)n.d为未检出
实施例2发酵腐竹豆清液对肠道短链脂肪酸及有益菌丰度的影响
1.试验方法
首先，体外模拟胃-小肠消化系统，模拟发酵腐竹豆清液摄入动物体内后在胃肠的过程。具体方法是：以1mol/L HCl调节发酵腐竹豆清液的pH值至2.0-3.0，随后添加模拟胃液(含72g/L胃蛋白酶)，于37℃，150rpm条件下胃消化2小时；随后以1mol/L NaHCO3调节胃消化产物pH至6.5，添加模拟小肠液(含10g/L胰蛋白酶和2g/L胆汁酸盐)，再以1mol/L NaOH调节pH至7.4，于37℃，150rpm条件下小肠消化2小时。
接着，将经过胃-小肠消化后的发酵腐竹豆清液进行模拟结肠处理，模拟结肠液的配制参考自文献(Sánchezpatán,et al.,2012)，模拟结肠液中添加的粪便菌悬液由来自健康人的新鲜粪便在厌氧条件下制得。发酵腐竹豆清液的添加量为50-60％的结肠液容积，模拟结肠处理的条件为：37℃，厌氧条件下静置培养48小时。
同时还进行以下对照试验：
(1)植物乳杆菌组为取活菌数为108CFU/mL的植物乳杆菌，以6％的接种量接种到与实验组相同体积的无菌PBS缓冲液中，然后按照与实验组相同的实验条件进行模拟胃-小肠-结肠消化过程。
(2)未发酵腐竹豆清液组为未发酵的腐竹豆清液原液按上述方法进行模拟消化试验。
(3)等体积BCM培养基按上述方法进行处理作为空白。
取模拟结肠液消化后的上清液，经50％的浓硫酸酸化，后经乙酸乙酯萃取，采用气相色谱法测定短链脂肪酸含量，色谱柱为安捷伦DB-FFAP。气相色谱的参数设定如下：上样量1μL，进样口温度250℃，压力13.6psi，分流比10:1，氮气作为载气；DB-FFAP毛细色谱柱，恒定流速1mL/min，色谱柱耐受温度为250℃；恒温箱升温程序106℃，3min，10℃/min升至170℃，70℃/min升至240℃维持3min，总时长13.4min；FID检测器温度250℃，空气：氮气＝400:40，以氮气进行尾吹。
另取模拟结肠液消化后的残渣，采用DNA提取试剂盒对肠道菌的总DNA进行提取，以双歧杆菌(Bifidobacterium)、瘤胃球菌(Ruminococcus)、丁酸产生菌、乳杆菌(Lactobacillus)、艾克曼菌(Akkermansia)以及大肠埃希氏菌的特定引物(表2)对目的基因进行定量PCR扩增。以16S rDNA为内参，使用TaKaRa SYBR Premix Ex TaqTMⅡ荧光定量试剂盒对提取的DNA进行定量扩增，在qTOWER 2.0 PCR System进行PCR扩增反应。反应程序为：首先在95℃预变性30s，其次进行40个循环的扩增：95℃，5sec；53℃，30sec；72℃，30sec。每次循环最后一步采集荧光。溶解曲线温度范围为60℃-95℃，每20sec升温1℃，采用2–ΔΔCt的方法对肠道微生物进行相对定量。
表2用于检测肠道菌相关基因的引物
2.试验结果
从表3可知，发酵腐竹豆清液经模拟消化道处理后，模拟结肠液中的乙酸、丙酸、丁酸、异戊酸和戊酸含量相比空白组均显著性增加(P＜0.05)；未发酵腐竹豆清液组中的丙酸、丁酸含量相比空白组显著性增加(P＜0.05)；而植物乳杆菌组中只有乙酸含量相比空白组显著性增加(P＜0.05)。综合比较发酵腐竹豆清液组明显比未发酵腐竹豆清液组和植物乳杆菌处理组短链脂肪酸含量高，以上结果说明，发酵处理后的腐竹豆清液对肠道短链脂肪酸的产生具有更好地促进作用。
肠道短链脂肪酸SCFAs水平能够指示肠道微生物种群的健康水平(Gijs D B etal 2013，Gil-Sánchez I et al 2017)。SCFAs含量的增加能够降低肠道的pH值，有利于益生菌的生长(Gil-Sánchez I et al 2018)。丙酸和丁酸能够促进动物抗菌肽基因表达,增加抗菌肽分泌，从而增强肠道免疫屏障，起到保护肠道作用(Sunkara L T et al 2012)。SCFAs也能够调节机体对于矿物质的吸收和电解质的平衡，乙酸、丙酸、丁酸能够减少Na+、K+和Cl-的分泌，从而维持机体的电解质稳态和肠道微生物稳态(陈福等2019)。
表3不同处理组对结肠中短链脂肪酸含量的影响(μmol/L)
注：(1)同一列不同角标字母的数据具有显著差异(P＜0.05)
从表4可知，经模拟消化道处理后，发酵腐竹豆清液组均具有较高的有益菌(双歧杆菌、瘤胃球菌、丁酸菌、乳杆菌及艾克曼菌)表达量，而有害菌(大肠埃希氏菌)的表达量很低，其在促进有益菌的产生及调节肠道菌群失调方面的效果要明显优于未发酵的腐竹豆清液和植物乳杆菌。
表4不同处理组对肠道有益菌、有害菌的相对表达量
注：(1)同一列不同角标字母的数据具有显著差异(P＜0.05)
实施例3微胶囊的制备及其缓释效果
由于发酵腐竹豆清液在经过胃-小肠消化系统时，活性成分很容易在复杂酸碱条件下被消化系统中的各种酶破坏，为了使发酵腐竹豆清液能更好发挥作用，我们对实施例1制备的发酵液进行了以下处理：
(1)将发酵液浓缩后真空冷冻干燥；
(2)采用化学交联法制备微胶囊：称取2g海藻酸钠溶于100mL蒸馏水中，制成浓度为2％的海藻酸钠溶液，再向海藻酸钠溶液中加入6g发酵腐竹豆清液冻干粉，持续搅拌至充分混匀。称取7.5g壳聚糖及15g CaCl2于500mL 1％的乙酸水溶液中，60℃恒温水浴并搅拌混匀，配置成壳聚糖浓度为1.5％、CaCl2浓度为3％的壳聚糖-CaCl2溶液。向壳聚糖-CaCl2混合溶液中滴加NaOH溶液，同时用注射器将含有发酵腐竹豆清液的海藻酸钠溶液匀速滴入壳聚糖-CaCl2混合溶液中，发生交联反应，过滤，干燥，即得包埋发酵腐竹豆清液的微胶囊。
接着，对制备的微胶囊进行以下检测，步骤如下：
(1)称取50mg发酵腐竹豆清液冻干粉溶解在100mL蒸馏水里，配制成一定质量浓度(50mg/100mL)的标准母液，采用紫外可见分光光度计进行全波段扫描，确定其最大吸收波长为225nm。
(2)分别取3.2mL、4.8mL、6.4mL、9.6mL、12.8mL、19.2mL、25.6mL母液定容至100mL容量瓶中，在225nm下测定其吸光度，绘制标准曲线(图1)，吸光值(x)与浓度(y)的关系式为y＝1.2793x+0.0027。
(3)取50mg微胶囊进行体外模拟胃-小肠消化，将消化各阶段的溶液在225nm下测定其吸光值Ai，胃消化过程以未加微胶囊的胃液为对照，小肠消化过程以未加样品的小肠液为对照，将Ai-A对照代入标准曲线得到释放出来的发酵腐竹冻干粉的含量，将50mg微胶囊以相同的消化体积的蒸馏水浸泡，以蒸馏水为对照，经彻底溶胀捣碎研磨后测定50mg微胶囊中所含发酵腐竹冻干粉的含量，测定微胶囊芯材冻干粉的释放率。
结果见图2，0-120min是模拟胃消化，120-240min模拟的是小肠消化。由图2可知0-30min内，微胶囊中的发酵腐竹豆清液快速释放，释放率迅速升至55.15％，之后释放率缓慢升高，直到胃消化结束时释放率缓慢升至59.36％。当进入肠消化时，释放率先快速升高再缓慢升高，可能与消化环境如pH或酶的改变有关。消化至180min时，释放率至78.62％，之后缓慢上升，肠消化结束后释放率达到82.90％。
结果表明，通过微胶囊包埋减少了发酵腐竹豆清液冻干粉与胃液、肠液的接触，减缓了在胃、小肠中的释放，有利于更多的成分进入结肠发挥活性。
由以上实施例可知，本发明制备的发酵腐竹豆清液对肠道短链脂肪酸以及有益菌的产生具有促进作用，对有害菌具有抑制作用，从而能促进动物肠道健康，可作为膳食补充剂在食品领域有潜在的利用价值，并且可作为饲料添加剂，在畜牧养殖业中具有广阔的应用前景。本发明还能够提高腐竹加工过程中的副产物利用率，减少浪费，降低环境污染，具有很好的经济与社会效益前景。
Claims (1)
1.一种促进肠道健康的膳食补充剂制备方法，其特征在于包括以下步骤：
S1：制备发酵腐竹豆清液：
将腐竹豆清液经高温灭菌后接种植物乳杆菌(Lactobacillus plantarum)菌液，然后在35-40℃恒温条件下静态发酵2-5天，所述植物乳杆菌菌液的接种量为腐竹豆清液体积的6％，菌液中的活菌数为108CFU/mL，发酵促进了结合型糖苷的水解，使苷元型的大豆异黄酮含量增加，所述发酵腐竹豆清液能促进肠道短链脂肪酸以及有益菌双歧杆菌、瘤胃球菌、丁酸菌、乳杆菌及艾克曼菌的产生，抑制有害菌大肠埃希氏菌的产生，从而促进动物肠道健康；
S2：制备微胶囊制剂：
(1)将所述的发酵腐竹豆清液浓缩后真空冷冻干燥，得冻干粉；
(2)配制浓度为2％的海藻酸钠溶液和壳聚糖浓度为1.5％、氯化钙浓度为3％的壳聚糖-CaCl2混合溶液；
(3)向海藻酸钠溶液中加入6％的发酵腐竹豆清液冻干粉，搅拌混匀；
(4)向壳聚糖-CaCl2混合溶液中滴加NaOH溶液，同时用注射器将步骤(3)得到的溶液匀速滴入壳聚糖-CaCl2混合溶液中，发生交联反应，过滤，干燥，即得包埋发酵腐竹豆清液的微胶囊。
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| WO2018103118A1 (zh) * | 2016-12-07 | 2018-06-14 | 深圳先进技术研究院 | 一种发酵型大豆乳清饮料及其制备方法 |
| CN107307086A (zh) * | 2017-06-08 | 2017-11-03 | 青岛科海生物有限公司 | 一种β‑葡萄糖苷酶在豆奶中的应用方法 |
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