CN115216501A - 一种通过固态复合发酵花生壳制备高品质膳食纤维的方法 - Google Patents
一种通过固态复合发酵花生壳制备高品质膳食纤维的方法 Download PDFInfo
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Abstract
本发明提供了一种通过固态复合发酵花生壳制备高品质膳食纤维的方法，涉及一种通过酶催化水解及固态发酵花生壳制备高品质膳食纤维的技术领域。本发明公开了一种筛选高产纤维素酶及半纤维素酶的霉菌和高产新型木聚糖酶的细菌的技术方案以及通过两种菌株的固态复合发酵提升花生壳中高品质膳食纤维的比例的方法。菌株包括：棒曲霉（Aspergillus clavatus）MZ211和类芽孢杆菌（Paenibacillus sp.）B1709，分别于2021年4月22日及2017年11月8日保藏于中国微生物菌种保藏委员会普通微生物中心（CGMCC），保藏编号分别为CGMCC NO.22410，CGMCC NO.14870。其特征在于本发明所涉及及产品属性以特定区域、特定对象筛选的高性能菌种提升特定对象的特定品质。本发明对开发一种利用花生壳制备高品质膳食纤维的方法具有十分重大的意义。
Description
技术领域
本发明涉及一种通过酶催化水解及固态发酵花生壳制备高品质膳食纤维的方法。
背景技术
近年来，随着食品工业的迅猛发展，食品加工的精度不断提高。人们膳食结构中膳食纤维的含量下降，导致各种不健康饮食引起的亚健康疾病普遍发生，预防这类疾病已成为食品、营养和流行病学领域的研究热点。许多研究表明，合理摄入膳食纤维可以预防亚健康疾病的发生或者降低患病风险，如肾结石、炎症、结肠癌和其他癌症、肥胖和心血管疾病等。此发现使得膳食纤维相关疾病流行病学以及降低疾病风险的潜在生理机制的研究迅速发展，同时也使得公众和食品行业迅速接受了膳食纤维作为健康饮食中有益的特殊营养位置。
目前，膳食纤维制备的方法主要分为三类：物理、化学和生物技术方法，也有综合以上手段共同处理的。其中生物技术法分为酶法和微生物发酵法。酶法是在调整酶解反应所需的最适pH值后，向原料中加入蛋白酶、纤维素酶、糖化酶、半纤维素酶等生物酶，通过控制反应条件，降解原料中的非纤维成分和纤维素、半纤维素等IDF成分，生成小分子单糖等SDF成分。酶法提取DF效率高，且制得的DF生理活性损失较小，色泽浅，纯度高，但因酶制剂生产成本较高，实际操作方面还无法广泛推广。微生物发酵法是一种相对安全，有效且低成本的方法。它利用淀粉酶、纤维素酶和自身分泌的其他酶系消耗原材料中的淀粉和蛋白质，疏松结构，以使不溶性大分子如纤维素和半纤维素更容易降解，IDF转化为SDF，并达到提高SDF得率的目的。
花生是重要的食用植物油原料。目前，花生壳仅有一小部分被用于制造人造板和动物饲料，大多数用来作燃料或弃去，造成了资源浪费。花生壳中富含膳食纤维，一般为65%以上，价格非常低而且很容易获得。因此开发一种高品质膳食纤维新型的制备方法具备十分重大的意义。
本发明所涉及在自行筛选的高产纤维素酶及半纤维素酶的霉菌及产新型木聚糖酶的细菌等，通过使用两种菌株的固态复合发酵配合有效提升处理对象中的高品质膳食纤维的比例。发明所涉及产品属性以特定区域、特定对象筛选的高性能菌种提升特定对象的特定品质为基本特点，这也是其区别于其它同类产品的一个主要特征。
发明内容
本发明的目的在于提供通过酶催化水解及固态发酵花生壳制备高品质膳食纤维的方法。
为达到上述目的，本发明的技术方案筛选提供了一种可实现较好利用花生壳进行发酵产生高品质膳食纤维的霉菌及细菌。分别经中国科学院微生物研究所鉴定为棒曲霉MZ211(Aspergillus clavatus MZ211) (该菌于2021年4月22日保藏于中国普通微生物菌种保藏管理中心，地址为：北京市朝阳区北辰西路1 号院3号中国科学院微生物研究所，保藏编号为：CGMCC NO.22410)以及类芽孢杆菌B1709(Paenibacillus sp.B1709)(该菌于2017年11月8日保藏于中国普通微生物菌种保藏管理中心，地址为：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为：CGMCC NO.14870)。
本发明涉及的具体制备方法，主要包括以下步骤：
（1）高产多种大分子降解酶及发酵适应性微生物的筛选
将筛选到的菌株分别进行平板初筛和发酵复筛。其中平板初筛培养基分别为：①木聚糖酶筛选培养基：榉木木聚糖1.0%，牛肉蛋白胨0.3%，酵母浸膏0.2%， KH2PO4 0.6%，K2HPO4 0.15%，MgSO4·7H2O 0.05%，FeSO4·7H2O 0.001%，琼脂2.0%，pH 6.0。121℃下杀菌20min。②淀粉酶筛选培养基：牛肉膏0.3%，氯化钠0.5%，胰蛋白胨1.0%，琼脂粉2.0%，可溶性淀粉2.0%，pH自然，121℃下杀菌20min。③蛋白酶筛选培养基：牛肉膏0.3%，氯化钠0.5%，胰蛋白胨1.0%，琼脂粉2.0%，脱脂奶粉1.5%，pH自然，121℃下杀菌20min。摇瓶复筛培养基：花生壳粉（干燥，过65目筛）4.0%，胰蛋白胨1.0%，酵母浸粉0.6%，NaNO3 0.4%，KH2PO4 0.2%，K2HPO4 0.1%，MgSO4·7H2O 0.05%，FeSO4·7H2O 0.001%， pH6.0。121℃下杀菌20min。取发酵液分别进行淀粉酶、木聚糖酶及蛋白酶的测定。
（2）发酵微生物制备
霉菌孢子菌悬液制备：用铂环收集真菌孢子，悬浮于无菌水中，调整浓度约为106~107个孢子/mL。将10 mL（10%左右）悬浮液（106~107孢子/mL）接种到100 mL无菌种子培养基中，然后，将上述用悬浮液接种的培养基在150 r/min，28 ℃下培养2 d，发酵完成后的液体培养基，过滤掉菌丝体，制成种子液备用；
细菌种子发酵液制备：挑取入选的斜面菌种接入种子培养基，37℃、180r/min培养14h，制成种子液备用。
（3）高品质花生壳膳食纤维的制备
接种两种微生物种子液各50mL（共100mL）种子液到1kg固体发酵培养基（花生壳粉（粗粉20-40目）40.0 g，牛肉蛋白胨10.0 g，酵母浸膏6.0 g，KH2PO4 2.0 g，K2HPO4 1.5 g，MgSO4·7H2O 0.5 g，FeSO4·7H2O 0.01 g，自然pH，补水至1000 mL）中，30℃、150 r/min培养4 d后，取发酵液过滤，10000 r/min离心10 min，沉淀物冷冻干燥，粉碎得到花生壳不溶性膳食纤维（IDF），收集上清液，添加95%乙醇（上层清液量的四倍），4 ℃下静置12 h，10000r/min离心10 min，将沉积物冷冻干燥并粉碎获得高活性花生壳可溶性膳食纤维（SDF），计算SDF得率。
附图说明
说明书附图共三张，包括：
（1）图1 SDF的理化性质；
（2）图2 SDF对葡萄糖吸附能力；
（3）图3 SDF对胆固醇吸附能力。
具体实施方式
本发明以下结合具体实例作进一步说明，但并不是限制本发明。
实施例1 利用两菌株复合发酵花生壳固态发酵制备高品质膳食纤维的工艺
接种100 mL种子液到1kg发酵培养基中，30℃、150 r/min培养4 d后，取发酵液过滤，10000 r/min离心10 min，沉淀物冷冻干燥，粉碎得到花生壳不溶性膳食纤维（IDF），收集上清液，添加95%乙醇（上层清液量的四倍），4 ℃下静置12 h，10000 r/min离心10 min，将沉积物冷冻干燥并粉碎获得玉米芯可溶性膳食纤维（SDF），计算SDF得率。
实施例2 膳食纤维高活性验证性试验
（1）考察原料中直接提取的SDF（相同含水量花生壳，加入耐高温α-淀粉酶，95 ℃下孵育30 min。随后，在60 ℃下，添加适量的糖化酶反应30 min。然后，将蛋白酶添加到溶液中30 min），用相同的方法获得花生壳可溶性膳食纤维（B-SDF）和复合发酵制备的SDF（F-SDF）的溶解度、持水力、持油力。
溶解度是指物质可以溶解的程度，这是SDF的重要参考指标。实验结果表明（见说明书附图图1），发酵处理明显提高了初始溶解度。SDF的溶解度从0.43 g/g增加到0.580g/g，提高了34.88%（其中B-SDF代表处理前的膳食纤维（空白对照）；B-SDF代表发酵后的膳食纤维）。据报道，SDF的多孔结构会导致其吸水膨胀。
持水力（WHC）定义为在施加外力（例如离心力）后，已知重量的水状胶体保留的水量，其特征是结合力强。高WHC的膳食纤维可防止食物收缩，并可改变某些食物的粘度，是膳食纤维良好功能特性的体现。由说明书附图图1可知，通过发酵处理，WHC从6.13g/g增加到14.92g/g。有研究发现具有良好WHC的膳食纤维归因于多糖的亲水基团，也与SDF的含量、粒径、表面性质和来源等因素密切相关。
膳食纤维保留油脂的能力在食品应用中很重要，例如，具有高OHC的膳食纤维可稳定高脂食品和乳品。由说明书附图图1可知，F-SDF的持油力比B-SDF的持油力高，经过发酵处理，OHC从5.21g/g增加到7.13g/g，这可能是由于结构经过发酵后变得更加疏松多孔，此外，也有可能是SDF中的***木聚糖，果胶，***半乳聚糖等，由于它们对脂质物质的强亲和力，可能有助于SDF吸附和清除饱和脂肪和不饱和脂质物质。高OHC是膳食纤维的重要特征，因为这种能力可能会干扰膳食中脂质的肠道吸收，从而有助于控制体重和异常血脂状况。
（2）膳食纤维吸收葡萄糖的能力
高品质的膳食纤维可以延迟或减少胃肠道中葡萄糖的消化吸收，这在控制血糖方面起着重要作用。因此分别考察了B-SDF和F-SDF对葡萄糖的吸附能力，结果如说明书附图图2所示。经过对比可以看出，经过复合发酵的SDF可以显着提高葡萄糖吸附能力，F-SDF对葡萄糖的吸附量为2910.36µmol/g，比B-SDF（2330.84 µmol/g）提高了24.86%。结合其结构分析结果，F-SDF的葡萄糖吸附能力提高可能是因为发酵产生的纤维素酶和木聚糖酶降解纤维表面，使纤维结构松散，孔隙增大，使葡萄糖分子更容易被吸到纤维中。而未经发酵的膳食纤维结构致密，大部分关键功能基团被纤维的内部结构包围，导致其无法发挥有效作用。
（3）胆固醇吸附能力
pH值是影响膳食纤维胆固醇吸附能力的重要因素，因此选用pH 2.0（模拟胃环境）和pH 7.0（模拟小肠环境）下，分别考察B-SDF和F-SDF对胆固醇的吸附量，结果见说明书附图图3。pH 2.0条件下，B-SDF的胆固醇吸附量为3.14 mg/g，相比于B-SDF，F-SDF的胆固醇吸附量为7.25mg/g，提高了1.31倍；pH 7.0条件下，B-SDF与F-SDF的胆固醇吸附量分别为8.45mg/g和13.57mg/g，提高了60.59%。显然，pH 7.0的CAC值高于pH 2.0的CAC值，表明F-SDF的CAC在模拟小肠环境中比在模拟胃环境中更强。结合扫描电镜图，F-SDF吸收胆固醇的能力高于B-SDF，这可能是由于膳食纤维结构松散和比表面积的增加，导致脂溶性物质更容易渗透到膳食纤维内部。
Claims (5)
1.一种通过固态复合发酵花生壳制备高品质膳食纤维的方法，其特征在于，自行筛选出高性能菌种进行固态复合发酵制备花生壳中的高品质膳食纤维。
2.根据权利要求书1所述，两种高性能菌种，其特征在于，分别为棒曲霉（Aspergillus clavatus）MZ211和类芽孢杆菌（Paenibacillus sp.）B1709，分别于2021年4月22日及2017年11月8日保藏于中国微生物菌种保藏委员会普通微生物中心（CGMCC），保藏编号分别为CGMCC NO.22410，CGMCC NO.14870。
3.根据权利要求书1或2所述的筛选高性能菌种，其特征在于，包括以下步骤：
（1）高产多种大分子降解酶及发酵适应性微生物的筛选
平板初筛：
①木聚糖酶筛选培养基：榉木木聚糖1.0%，牛肉蛋白胨0.3%，酵母浸膏0.2%， KH2PO40.6%，K2HPO4 0.15%，MgSO4·7H2O 0.05%，FeSO4·7H2O 0.001%，琼脂2.0%，pH 6.0，121℃下杀菌20min；
②淀粉酶筛选培养基：牛肉膏0.3%，氯化钠0.5%，胰蛋白胨1.0%，琼脂粉2.0%，可溶性淀粉2.0%，pH自然，121℃下杀菌20min；
③蛋白酶筛选培养基：牛肉膏0.3%，氯化钠0.5%，胰蛋白胨1.0%，琼脂粉2.0%，脱脂奶粉1.5%，pH自然，121℃下杀菌20min；
摇瓶复筛：
摇瓶复筛培养基：花生壳粉（干燥，过65目筛）4.0%，胰蛋白胨1.0%，酵母浸粉0.6%，NaNO30.4%，KH2PO4 0.2%，K2HPO4 0.1%，MgSO4·7H2O 0.05%，FeSO4·7H2O 0.001%， pH6.0，121℃下杀菌20min；
（2）发酵微生物制备
霉菌孢子菌悬液制备：用铂环收集真菌孢子，悬浮于无菌水中，调整浓度约为106~107个孢子/mL；将10 mL（10%左右）悬浮液（106~107孢子/mL）接种到100 mL无菌种子培养基中，然后，将上述用悬浮液接种的培养基在150 r/min，28 ℃下培养2 d，发酵完成后的液体培养基，过滤掉菌丝体，制成种子液备用；
细菌种子发酵液制备：挑取入选的斜面菌种接入种子培养基，37℃、180r/min培养14h，制成种子液备用。
4.根据权利要求书1所述的固态复合发酵制备花生壳中的高品质膳食纤维，其特征在于，1:1接种两种菌株种子液（共100mL）到1kg由花生壳制得的固体发酵培养基中, 30℃、150 r/min培养4 d后，取发酵液过滤，10000 r/min离心10 min，沉淀物冷冻干燥，粉碎得到花生壳不溶性膳食纤维（IDF），收集上清液，添加95%乙醇（上层清液量的四倍），4 ℃下静置12 h，10000 r/min离心10 min，将沉积物冷冻干燥并粉碎获得玉米芯可溶性膳食纤维（SDF）。
5.根据权利要求书1或4所述的花生壳中的高品质膳食纤维，其特征在于，膳食纤维高活性验证性试验，包括以下方面：
（1）考察原料中直接提取的SDF（相同含水量花生壳，加入耐高温α-淀粉酶，95 ℃下孵育30 min，随后，在60 ℃下，添加适量的糖化酶反应30 min，然后，将蛋白酶添加到溶液中30 min），比较用相同的方法从原料中直接获得花生壳可溶性膳食纤维（B-SDF）和复合发酵制备的SDF（F-SDF）的溶解度、持水力、持油力，实验结果表明F-SDF的溶解度、持水力和持油力分别相比于B-SDF均有所增加；
（2）膳食纤维吸收葡萄糖的能力
分别考察B-SDF和F-SDF对葡萄糖的吸附能力，结果表明，经过复合发酵的SDF可以显着提高葡萄糖吸附能力，F-SDF对葡萄糖的吸附量为2910.36µmol/g，比B-SDF（2330.84 µmol/g）提高了24.86%；
（3）胆固醇吸附能力
选用pH 2.0（模拟胃环境）和pH 7.0（模拟小肠环境）下，分别考察B-SDF和F-SDF对胆固醇的吸附量（CAC），结果表明，PH 2.0条件下，相比于B-SDF的胆固醇吸附量为3.14 mg/g，F-SDF的胆固醇吸附量为7.25mg/g，提高了1.31倍；pH 7.0条件下，B-SDF与F-SDF的胆固醇吸附量分别为8.45mg/g和13.57mg/g，提高了60.59%；pH 7.0的CAC值高于pH 2.0的CAC值，说明F-SDF的CAC在模拟小肠环境中比在模拟胃环境中更强。
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