CN112075629B - 通过益生菌发酵提高南非醉茄提取物抗氧化活性的方法 - Google Patents
通过益生菌发酵提高南非醉茄提取物抗氧化活性的方法 Download PDFInfo
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- CN112075629B CN112075629B CN202011014149.7A CN202011014149A CN112075629B CN 112075629 B CN112075629 B CN 112075629B CN 202011014149 A CN202011014149 A CN 202011014149A CN 112075629 B CN112075629 B CN 112075629B
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- withania somnifera
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Abstract
本发明属于微生物发酵技术领域，具体涉及一种利用乳酸菌发酵方案来提高南非醉茄提取物抗氧化活性的方法，还涉及通过上述的方法发酵获得的提取物在抗氧化产品中的应用，以及在改善食品风味中的应用。本发明所提供的提高南非醉茄提取物抗氧化活性的方法主要是通过采用植物乳杆菌、干酪乳杆菌、嗜酸乳杆菌、发酵乳杆菌来发酵南非醉茄提取物来达到上述目的的。本发明通过实验，结果证明利用乳酸菌发酵南非醉茄提取物之后，提取物中总酚含量较发酵之前增加12.3~36.1%、DPPH自由基清除能力提高了48.65~59.7%、ABTS自由基清除能力提高了1.2~6.4%、铁离子还原力FRAR值增幅为9.1~44.1%、总抗氧化能力9.7~36.7%。
Description
技术领域
本发明属于微生物发酵技术领域，具体涉及一种利用乳酸菌发酵方案来提高南非醉茄提取物抗氧化活性的方法及其应用。
背景技术
南非醉茄是一种传统草药，有多种治疗用途和药理活性，是治疗神经疾病的草药复方制剂“Mentat”的主要组分之一。其对于小鼠的脑代谢具有改善作用。南非醉茄提取物粉末至少含睡茄内酯糖苷和寡糖及游离的withaferinA；能有效的提高认知及学习能力，减轻压力，具有免疫调节的作用，并且能提高抗氧化防御酶活性。如超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化酶。
南非醉茄因其较好的生理活性广泛应用于功能性食品的开发，然而目前有关南非醉茄发酵的研究报道较少。Che’rifRabhi等人使用真菌Beauveria bassiana ATCC 7159发酵南非醉茄, 余甘子和假马齿苋的提取物混合物，发酵促进了浓混合液的流化，去除了余甘子提取物中的葡糖苷，增加了没食子酸的含量。此外，发酵的提取物没有任何毒性，且具有较好的抗血管生成的效力，最终开发出一款抗血管增生的商业化营养制剂。但是关于乳酸菌发酵后南非醉茄提取物是否能提高其原有的活性比如抗氧化活性等，以及南非醉茄提取物对于乳酸菌是否有抑制作用，目前并没有相关的文献披露。
发明内容
为了解决上述的技术问题，本发明提供了一种利用乳酸菌发酵南非醉茄提取物以便获得总酚含量更高、抗氧化活性更强的产品的方法。
为了解决上述的技术问题，本发明利用乳酸菌发酵南非醉茄提取物，并提供了一种提高南非醉茄提取物抗氧化活性的发酵方法，并且通过该方法获得的产品还能显著的改善食品的风味。本发明所提供的一种提高南非醉茄提取物抗氧化活性的方法，最大的特点是，利用乳酸菌发酵南非醉茄提取物以使提取物中总酚含量显著增加、抗氧化活性提高。
本发明中的乳酸菌为：植物乳杆菌、干酪乳杆菌、嗜酸乳杆菌、发酵乳杆菌，它们为均等份；
乳酸菌发酵南非醉茄提取物的具体步骤如下：
（1）南非醉茄提取物制备：取南非醉茄根粉，将其粉碎至粒径70~350μm，按料液比1:10～1:50的比例加水，采用索式萃取或超声萃取或煮沸的方法，获得南非醉茄提取物液体；然后通过喷雾干燥/旋转蒸发将提取物液体转化为粉体或浸膏；
（2）发酵基质制备：称（1）中所获得的的粉体或浸膏，配制含南非醉茄提取物的发酵基质，发酵基质中提取物含量为0.5%~4%，葡萄糖含量为0.5%~5%，蛋白胨含量为0.2~3%；基质于115℃~121℃灭菌10 min~25 min，灭菌处理后备用；
（3）发酵种子液制备：将植物乳杆菌、干酪乳杆菌、嗜酸乳杆菌、发酵乳杆菌涂布于MRS固体板进行活化，挑取单菌落至MRS液体培养基中，30~37℃培养24h。继代两次后放大培养，24 h后将培养液离心去上清，然后用磷酸缓冲液冲洗菌体后重悬，得到浓缩种子液；
（4）发酵：将任意一种或多种组合的种子液，接种至发酵基质中，接种密度为104~107log CFU/mL，发酵温度30～37℃，发酵时间24~120h。
利用乳酸菌发酵南非醉茄提取物之后，提取物中总酚含量较发酵之前增加12.3~36.1% 、DPPH自由基清除能力提高了48.65~59.7%、ABTS自由基清除能力提高了1.2~6.4%、铁离子还原力FRAR 值增幅为9.1~44.1%、总抗氧化能力9.7~36.7%。
通过上述的方法获得的南非醉茄提取物在抗氧化中的应用，尤其是在抗氧化产品中的应用，以及在改善食品风味中的应用是本发明所要重点保护的范围。
上述的抗氧化产品为食品、化妆品或药品中的任一种；抗氧化产品可以是液体产品或固体产品或悬浊液、乳浊液产品或其它形式的产品，不限于产品的形式。比如产品为固体饮料、液体饮料中的任一种。
本发明的有益效果在于：
（1）利用乳酸菌发酵提高了南非醉茄提取物中的总酚含量和抗氧化活性；
（2）改善了南非醉茄提取物的原有风味，从气相离子色谱的检测结果来看，发酵前后风味物质图谱发生较大变化，发酵后醇类和酮类物质显著增加；
（3）拓宽了南非醉茄提取物的应用形式，丰富了以南非醉茄为主要原料的相关产品形式。
附图说明
图1为南非醉茄提取物发酵过程中菌量、pH、残糖和总酸的动态变化；A为菌量变化图；B为pH值变化图；C为残糖含量变化图；D为总酸含量变化图；
图2为南非醉茄提取物发酵前后总黄酮和总酚含量图；UF，未发酵；F1，发酵菌种为植物乳杆菌（Lactobacillus plantarum）；F2，发酵菌种为干酪乳杆菌 （L. casei）；F3，发酵菌种为嗜酸乳杆菌（L. acidophilus）；F4，发酵菌种为发酵乳杆菌（L. fermentum）；FMIX，发酵菌种为以上菌种组合（1:1:1:1）；
图3为南非醉茄提取物发酵前后抗氧化活性的变化图；UF，未发酵；F1，发酵菌种为植物乳杆菌（L. plantarum）；F2，发酵菌种为干酪乳杆菌 （L. casei）；F3，发酵菌种为嗜酸乳杆菌（L. acidophilus）；F4，发酵菌种为发酵乳杆菌（L. fermentum）；FMIX，发酵菌种为以上菌种组合（1:1:1:1）；
图4 为南非醉茄提取物发酵前后风味组分的对比差异谱图；以未发酵样品为参照（左），对比显示样品中所有挥发性物质在不同样品中的差异情况，红色代表该物质在该样品中浓度高于参照样品，而蓝色则代表低于参照样品；
图5为南非醉茄提取物发酵前后风味组分的Gallery Plot指纹谱图；UF为未发酵处理；F为发酵处理，每个处理三个平行。
具体实施方式
为了能使本领域技术人员更好的理解本发明，现结合具体实施方式对本发明进行更进一步的阐述。
实施例1
乳酸菌发酵南非醉茄提取物的具体步骤如下：
（1）取喷雾干燥的南非醉茄提取物粉2 g，葡萄糖2.5 g，蛋白胨1.25 g，蒸馏水100mL；混合均匀后在115℃灭菌15 min，冷却备用；
南非醉茄提取物制备：取南非醉茄根粉，将其粉碎至粒径70~350μm，以料液比1：12（质量体积比）的比例加入水，采用超声萃取的方法，获得南非醉茄提取物液体；然后通过喷雾干燥将提取物液体转化为粉体；
（2）分别取植物乳杆菌（L. plantarum）、干酪乳杆菌（L.
casei）、发酵乳杆菌（L. fermentum）、嗜酸乳杆菌（L. acidophilus）进行活化，继代2次后至培养箱中放大培养，其中植物乳杆菌、干酪乳杆菌、嗜酸乳杆菌培养温度为35℃，发酵乳杆菌培养温度为30℃。静置培养24 h后，于3000 rpm 离心10 min，弃上清液，用磷酸缓冲液（0.1M，pH=6.8）洗涤三次，用无菌水重悬调整至密度108log CFU/mL备用；
（3）将制备的种子液按照植物乳杆菌（L. plantarum）、干酪乳杆菌（L. casei）、发酵乳杆菌（L. fermentum）、嗜酸乳杆菌（L. acidophilus）=1:1:1:1的比例接入含有南非醉茄提取物的基质中，35℃发酵72 h。
实施例2
南非醉茄提取物对于乳酸菌是否有抑制作用，具体实验如下：
在含有南非醉茄提取物的溶液中接种乳酸菌，采用不同发酵时期的菌量和其他代谢指标评价南非醉茄提取物对乳酸菌生长的影响。在2%南非醉茄提取物的基质中分别接种各乳酸菌及组合，通过对不同发酵时间（0 h，24 h，48 h，72 h）的菌量、残糖、pH和总酸的测定，判断南非醉茄提取物对乳酸菌生长的影响。
从附图1可以看出，乳酸菌可在含南非醉茄提取物的培养基中增殖生长，并且进行正常的代谢活动，说明南非醉茄提取物对乳酸菌无抑制作用。
实施例3
检测实施例1中的提取物在发酵前后的总黄酮和总酚的变化；具体的检测方法如下：
（1）总黄酮含量的测定
采用亚硝酸钠-硝酸铝法。将3.0 mL 不同处理的发酵液（稀释5倍）加入到25 mL比色管中, 然后加入1 mL的亚硝酸钠（5%，w/v），充分混匀后静置5 min，加入1 mL的三氯化铝（10%，w/v），混匀后25℃水浴6 min，加入10 mL氢氧化钠溶液（1 mol/L），最后补足蒸馏水至25 mL，充分混匀放置15 min。于510 nm测定吸光度值，以芦丁为标准品做标准曲线，不同处理发酵液中总黄酮含量以μg (rutin equivalent, RE)/mL 表示。
（2）总酚含量的测定
采用Folin–Ciocalteu测定方法。取0.5 mL不同不同处理的发酵液（稀释5倍），加入0.5 mL蒸馏水和2 mL Folin–Ciocalteu 试剂（稀释10倍，现用现配），混匀后反应5 min。加入2.0 mL碳酸钠溶液7.5%（w/v），黑暗条件下25℃水浴反应30 min。于765 nm 测定吸光度值，以没食子酸为标准品做标准曲线，不同处理发酵液中总多酚含量以μg (galic acidequivalent, GAE)/mL 表示。
从图2可以看出，南非醉茄提取物经过不同菌种发酵后，总黄酮含量总体上下降，降幅在3.0%~39.2%；总酚含量总体上增加，增幅在12.3~36.1%。
实施例4
检测实施例1中的提取物在发酵前后抗氧化性能的变化；具体的检测方法如下：
（1）DPPH自由基清除能力的测定
分别取1.0 mL不同处理的各样品溶液（稀释5倍），置10 mL离心管中，加入3.0 mL的DPPH溶液（0.008 mmol/L），室温避光反应30 min，同时以无水乙醇为空白，于517 nm处测定吸光度。Vc（0.1 mg/mL）作为阳性对照。按下列公式计算DPPH自由基清除率。
DPPH自由基清除率（%）= A0-(As-Ac)/ A0×100
式中，A0—1.0 mL蒸馏水+3.0 mL DPPH溶液的吸光度，
As—1.0 mL样品溶液+3.0 mL DPPH溶液的吸光度，
Ac—1.0 mL样品溶液+3.0 mL无水乙醇的吸光度。
（2）ABTS自由基清除能力的测定
分别取0.1 mL不同处理的各样品溶液（稀释5倍），加入0.1 mL无水乙醇，混匀，再加入0.8 mL的ABTS+溶液（0.1 mmol/L），充分混匀后静置6 min，同时以无水乙醇为空白，于734 nm处测定吸光度。Vc（0.1 mg/mL）作为阳性对照。按下列公式计算ABTS自由基清除率。
ABTS自由基清除率（%）= （A0-A)/ A0×100
式中，A0—0.2 mL蒸馏水+0.8 mL ABTS+溶液的吸光度，
A—0.2 mL样品溶液+0.8 mL ABTS+溶液的吸光度。
（3） 总抗氧化活性的测定
采用钼酸铵法进行测定。分别取1.0 mL不同处理的各样品溶液（稀释5倍），置10mL离心管中，加入3.0 mL试剂溶液（试剂溶液中含0.6 mol/L的硫酸，28 mmol/L的磷酸钠和4 mmol/L的钼酸铵），混合液于95℃水浴锅中加热150 min，冷却至室温，以蒸馏水代替样品的处理为空白，于695 nm处测定其吸光度。吸光度越高，表明抗氧化能力越强。Vc（0.1 mg/mL）作为阳性对照。
（4）铁离子还原法（FRAP）的测定
取不同处理的各样品溶液0.3 mL（稀释5倍），加入2.7 mL预热至37℃的FRAP工作液（含0.83 mmol/L的TPTZ、1.67mmol/L的FeCl3•6H2O、0.25mol/L的醋酸缓冲液），摇匀后放置10 min，于593 nm测其吸光度值，以无水乙醇代替样品加入FRAP工作液作为空白。根据所得吸光值，在标准曲线上求得相应FeSO4浓度，定义为FRAP值，其值越大，抗氧化活性越强，Vc（0.1 mg/mL）作为阳性对照。每个浓度做3次，求平均值。
FeSO4标准曲线的绘制：配制800 μmol/LFeSO4标准溶液，梯度稀释后分别得到400，200，100，50，25 μmol/L的溶液，加入FRAP工作液，按照上述步骤进行，根据所得OD值绘制标准曲线。
检测结果如附图3所示，从图3可以看出，利用乳酸菌发酵南非醉茄提取物之后，发酵液的各项抗氧化指标总体上呈增加趋势。其中，DPPH自由基清除能力提高了48.65~59.7%、ABTS自由基清除能力提高了1.2~6.4%、铁离子还原力FRAR 值增幅为9.1~44.1%、总抗氧化能力9.7~36.7%。其中，个别单菌表现出较好的增加效果，而有些菌种则不然，总体上混合菌发酵的效果优于单菌发酵。
实施例5
检测实施例1中的提取物在发酵前后风味物质组分的变化；具体的检测方法如下：
利用气相-离子迁移谱（FlavourSpec®风味分析仪）对南非醉茄提取物发酵前后挥发性物质的差异分析。取1g样品，置于20mL顶空瓶中，50℃孵育15分钟后进样500μL。
具体测定条件如下
顶空进样条件：顶空孵化温度50 ℃；孵化时间15 min；振荡加热；顶空进样针温度55 ℃；进样量500 μL，不分流模式；载气为高纯氮气；清洗时间0.5 min。
GC 条件：色谱柱温度60 ℃；运行时间为20 min；载气为高纯氮气； 流速初始为2.0 mL/min，保持2 min 后在18 min 内线性增至150 mL/min。
离子迁移谱单元条件：色谱柱MXT-5 15m ID:0.53mm；漂移管长度5 cm；管内线性电压500 V/cm；漂移管温度40 ℃；漂移气为高纯氮气；流速为150 mL/min；IMS 探测器温度45 ℃。
检测结果如附图4和5所示。当以未发酵的样品为参照差异对比时，如图4所示，发酵样品中对应的挥发性物质的浓度高低一目了然，红色越深，说明对应物质浓度比UF-NFSTW中浓度高的越多；蓝色越深则反之。
图5是两个样品挥发性物质组成的指纹谱图，可以看出在未发酵的样品中醛类及酯类物质相对含量更高，例如乙酸丁酯、乙酸乙酯、γ-羟基丁内酯、丁醛、戊醛、己醛、庚醛、壬醛、2-甲基丁醛、3-甲基丁醛、糠醛、苯乙醛等；而在发酵的样品中醇类和酮类物质的相对含量更高，例如3-羟基-2-丁酮、2,3,-丁二酮、2-丁酮、2-戊酮、环己酮、戊醇、3-甲基-3-丁烯醇等。
Claims (4)
1.一种提高南非醉茄提取物抗氧化活性的方法，其特征在于，所述的方法是利用乳酸菌发酵南非醉茄提取物以使提取物中总酚含量显著增加、抗氧化活性提高；
所述的乳酸菌为：植物乳杆菌、干酪乳杆菌、嗜酸乳杆菌、发酵乳杆菌，它们为均等份；
所述的乳酸菌发酵南非醉茄提取物的具体步骤如下：
（1）南非醉茄提取物制备：取南非醉茄根粉，将其粉碎至粒径70~350μm，按料液比1:10～1:50的比例加水，采用索式萃取或超声萃取或煮沸的方法，获得南非醉茄提取物液体；然后通过喷雾干燥/旋转蒸发将提取物液体转化为粉体或浸膏；
（2）发酵基质制备：称（1）中所获得的的粉体或浸膏，配制含南非醉茄提取物的发酵基质，发酵基质中提取物含量为0.5～4%，葡萄糖含量为0.5～5%，蛋白胨含量为0.2～3%；基质于115～121℃下灭菌10～25 min，灭菌处理后备用；
（3）发酵种子液制备：将植物乳杆菌、干酪乳杆菌、嗜酸乳杆菌、发酵乳杆菌涂布于MRS固体板进行活化，挑取单菌落至MRS液体培养基中，30~37℃培养24h；继代两次后放大培养，24 h后将培养液离心去上清，然后用磷酸缓冲液冲洗菌体后重悬，得到浓缩种子液；
（4）发酵：将任意一种或多种组合的种子液，接种至发酵基质中，接种密度为108 CFU/mL，发酵温度30～37℃，发酵时间24~120h。
2.通过权利要求1所述的方法获得的南非醉茄提取物在抗氧化中的应用。
3.通过权利要求1所述的方法获得的南非醉茄提取物在抗氧化产品中的应用。
4.如权利要求3 所述的应用，其特征在于，所述的抗氧化产品为化妆品或药品中的任一种。
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