CN114916676A - 一种含水苏糖和魔芋微粉的微囊化益生菌及其制备方法 - Google Patents
一种含水苏糖和魔芋微粉的微囊化益生菌及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种微囊化益生菌,具体公开一种含水苏糖和魔芋微粉的微囊化益生菌的制备方法,步骤如下:1)将菌悬液、水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液按质量比1:1~10:1~3:1~3:1~5混合,得到芯材混合液;2)将步骤(1)得到的芯材混合液与脱脂乳粉溶液和浓度为5%~20%的海藻酸钠溶液混合,得到混合液;3)将步骤(2)得到的混合液滴入氯化钙溶液中,形成微囊,微囊凝固后,过滤,除去水相,洗涤,得到微囊化益生菌。本发明制备的含水苏糖和魔芋微粉的微囊化益生菌产品货架期长,益生菌存活率高,环境抗逆性强。
Description
技术领域
本发明属于生物技术领域,具体涉及一种含水苏糖和魔芋微粉的微囊化益生菌及其制备方法。
背景技术
益生菌是人体肠道重要的生理菌,具有调节肠道菌群平衡、抑制有害菌、增强免疫力、抗癌等作用,被广泛应用于食品领域、医药领域以及畜牧领域。但益生菌对环境较为敏感,益生菌在生产、运输、销售和食用到胃肠道过程中,需经受一系列不良环境,导致益生菌活菌数大幅降低,影响其发挥益生效果。目前的益生菌产品主要有以下共性特点:(1)益生菌产品稳定性较差,易被空气中的氧气氧化,活性损失速率较快,导致货架期内活性降低,功效降低,不利于成品质量控制。(2)市场上销售的益生菌产品,经口服到达肠道前,已基本被胃酸和胆盐破坏,活性损失较大,难以发挥原有功效。另一方面,国内外正在通过生物技术开发高酸、高温耐受性的益生菌变异菌株,通过基因工程构造新菌株来提高益生菌稳定性的方法,但是这种方法需要大量的时间和较高的成本,同时还存在安全性争议,目前还没有产品得到应用。
针对益生菌产品活性保持时间短的技术问题,开发一种保持长时间活性的益生菌产品尤为重要。将益生菌包埋在微胶囊内可减轻环境中不良因素的影响,采用微胶囊制备技术的益生菌的抗逆性、稳定性大幅度提高,有利于其在肠道环境下功能的发挥,便于运输和长期保存。
本发明提供的一种含水苏糖和魔芋微粉的微囊化益生菌可显著提高益生菌在货架期的活菌含量,同时还能使大部分益生菌在肠道内释放,从而发挥有益效果。
发明内容
本发明的目的在于提供一种含水苏糖和魔芋微粉的微囊化益生菌及其制备方法。
基于上述目的,本发明采用如下技术方案:
本发明提供了一种含水苏糖和魔芋微粉的微囊化益生菌的制备方法,包括以下步骤:
(1)将菌悬液、水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液按质量比1:1~10:1~3:1~3:1~5混合,得到芯材混合液;
(2)将步骤(1)得到的芯材混合液与脱脂乳粉溶液和浓度为5%~20%的海藻酸钠溶液混合,得到混合液;
(3)将步骤(2)得到的混合液滴入氯化钙溶液中,形成微囊,微囊凝固后,过滤,除去水相,洗涤,得到微囊化益生菌。
优选地,步骤(1)中菌悬液的制备方法为:将益生菌于36℃条件下接种培养,发酵18h,得到益生菌发酵液。将益生菌发酵液离心,收集菌泥,菌泥经浓度为0.85%的无菌生理盐水洗涤2~3次后重悬于无菌水中,震荡混匀,得到菌悬液。所述离心的转速为1000~6000r/min,离心时间为3~20min。
优选地,步骤(1)中所述菌悬液中含有的益生菌为两歧双歧杆菌、长双歧杆菌、鼠李糖乳杆菌、罗伊氏乳杆菌、嗜酸乳杆菌中的至少一种。
优选地,步骤(1)所述菌悬液中每种益生菌的浓度均为107~1011CFU/mL。
优选地,步骤(1)中菌悬液、水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液的浓度分别为10%~20%、2%~5%、2%~10%、0.1%~1%。
优选地,步骤(2)中芯材混合液、脱脂乳粉溶液、海藻酸钠溶液地质量比为1:1~20:1~20,所述脱脂乳粉溶液的浓度为5%~20%。
优选地,步骤(3)中氯化钙溶液的浓度为1%~10%。
优选地,步骤(3)形成微囊的过程中需要搅拌,搅拌速度为200~1000r/min,搅拌时间为10~30min。
本发明还提供了一种由上述方法制备的含水苏糖和魔芋微粉的微囊化益生菌产品。
与现有技术相比,本发明取得的积极有益效果为:
(1)本发明以益生菌和具有显著益生活性的益生元为芯材,以海藻酸钠、脱脂乳粉、氯化钙的混合物为壁材,采用微胶囊技术制备得到了含水苏糖和魔芋微粉的微囊化益生菌,本发明制备的益生菌稳定性较高,存活率均在80%以上,常温保存60d后仍有78%以上的存活率,存储稳定性好,能够延长益生菌产品的货架期,有助于解决菌体不耐胃酸、不易保存的问题,使其益生功能得到有效发挥。
(2)水苏糖具有活性因子和良好的保护作用,能够提高益生菌的活性。
乳果糖能够改善肠道环境,使肠道变成酸性环境,有利于益生菌生长繁殖,抑制对人体有害细菌的生长繁殖,因此乳果糖可以对人体肠道的微生态有很好的调节作用。
低聚异麦芽糖属于多功能性寡糖,抑制肠道有害菌及腐败物质形成,提高机体免疫力。低聚异麦芽糖不会被胃和小肠吸收,而是直接进入大肠,被双歧杆菌优先利用,助其大量繁殖,系为双歧杆菌增殖因子;而肠内其它有害菌则不能利用,从而能抑制有害菌生长,促使肠道内微生态向良性循环调整。
魔芋微粉含有丰富的水溶性纤维,可以抑制肠胃吸收糖类,有降低血糖的作用;含有的葡萄甘露聚糖,能够阻止身体消化和吸收胆固醇,从而起到良好的降血脂和降胆固醇作用;含有的凝胶样成分,是一种具有抗癌防癌的化学物质,在进入人体后,会形成半透明膜衣,附着在肠壁上,阻止各种有害物质,起到抗癌防癌的功效。
本发明将水苏糖、乳果糖、低聚异麦芽糖和魔芋微粉加入益生菌中共同作为芯材,小分子低聚糖的加入一方面能提高微胶囊隔酸隔氧效果,保护益生菌免受胃酸侵蚀,另一方面溶解后小分子低聚糖还能作为益生菌的益生元,提高益生菌的活性。魔芋微粉不但具有保健功能,同时还能改善益生菌性能,延长其货架期。
(3)本发明微囊化益生菌的制备方法简单,易操作,制备的微囊化益生菌含有多种益生元活性组分,不添加任何化学合成物质,保证产品的安全性,可广泛应用于食品领域,具有显著的经济效益。
具体实施方式
(一)探究不同芯材组分对微囊化益生菌存活率的影响
为了探究不同芯材组分对微囊化益生菌存活率的影响,本发明进行了实施例1~实施例16实验,实施例1~实施例16实验的具体内容如下。
实施例1:
(1)将两歧双歧杆菌、长双歧杆菌、鼠李糖乳杆菌、罗伊氏乳杆菌、嗜酸乳杆菌于36℃条件下接种培养,发酵18 h ,得到益生菌发酵液。将益生菌发酵液以转速4000r/min离心5min,收集沉淀物,得到菌泥。将得到的菌泥用0.85%无菌生理盐水洗涤2~3次,将洗涤后的菌泥重悬于无菌水中,震荡混匀,得到每种菌浓度均为109 CFU/mL的菌悬液;
(2)将菌悬液、浓度为15%的水苏糖溶液、浓度为3%的乳果糖溶液、浓度为4%的低聚异麦芽糖溶液和浓度为0.4%的魔芋微粉溶液按质量比1:5:2:2:3混合,得到芯材混合液;
(3)将芯材混合液、浓度为8%的脱脂乳粉溶液和浓度为2%的海藻酸钠溶液按质量比1:5:5混合,得到混合液;
(4)将均匀挤压的混合液滴入浓度为2%的氯化钙溶液中,形成微囊,磁力搅拌器以转速400r/min搅拌15min,待微囊充分凝固后用蒸馏水冲洗3次,除去多余的钙离子和未被包埋的菌体,得到微囊化益生菌。
实施例2~实施例16的内容与实施例1基本相同,其不同之处在于:步骤(2)芯材混合液的组分不同,具体参见表1。
称取实施例1~16制备的微囊化益生菌,进行常温下保存、模拟胃液中处理和模拟肠液中处理后取样进行活菌计数,然后根据活菌数计算结果。同时,为了与本发明制备的含水苏糖和魔芋微粉的微囊化益生菌进行对比,本发明以实施例1步骤(1)中制备的菌悬液作为对比例,对菌悬液同步进行常温下保存、模拟胃液处理和模拟肠液处理的对比实验,实验结果如表1所示。
模拟胃液的配制:取稀盐酸16.4 mL,加水约800 mL,调pH到2.0,再加入胃蛋白酶10 g,搅匀后加水定容至1000 mL即可。
模拟肠液的配制:磷酸二氢钾6.8 g,加水500 mL溶解,用0.4 %的氢氧化钠调节pH至6.8,于120 ℃灭菌20 min;另取胰蛋白酶10 g,加适量无菌水使溶解,将两液混合后,加水定容至1000 mL即可。
微囊化益生菌在模拟胃液中处理的具体实验操作:取0.5 g微囊化益生菌加入到模拟胃液(4.5 mL)中,充分混匀15 s,在37 ℃条件下,以200 r/min在摇床中震荡处理30、60、90、120、150 min,分别离心并取样,计算活菌数及存活率。
微囊化益生菌在模拟肠液中处理的具体实验操作:取0.5 g微囊化益生菌加入到模拟肠液(4.5 mL)中,充分混匀15 s,在37 ℃条件下,在摇床中震荡处理30、60、90、120、150 min,分别离心并取样,计算活菌数及释放率。
所述存活率和释放率的计算公式为:
存活率(%)=A / B×100%;A表示处理后样品中的活菌数,单位CFU/mL;B表示未经处理前的活菌数,单位CFU/mL;
释放率(%)=V2 / V1×100%;V1表示未经处理前的活菌数,单位CFU/mL;V2表示经模拟肠液处理后,微胶囊破裂释放的细胞活菌数,单位CFU/mL。
由表1可以看出,未包埋的菌悬液中益生菌的存活率较低,常温保存60d后,存活率只有28.0%,经模拟胃液处理150min后存活率仅剩16.0%,益生菌被胃酸和胆盐破坏,活性损失较大。芯材只有益生菌的微胶囊,常温保存60d后,存活率为55.3%,与未包埋的益生菌相比存活率有明显提升,但经胃液处理150min后,存活率降至45.4%,说明只进行包埋的益生菌在低酸环境中仍难以存活,以致其在模拟肠液处理90min后,益生菌释放率仅为55.8%。而添加水苏糖、乳果糖、低聚异麦芽糖和魔芋微粉中的任意一种或几种与益生菌共同作为芯材时,益生菌的存活率和释放率均有明显提升,其中当同时添加水苏糖、乳果糖、低聚异麦芽糖和魔芋微粉时,常温保存60d后,存活率最高达90.0%,肠液释放率最高达93.0%,说明添加的水苏糖、乳果糖、低聚异麦芽糖和魔芋微粉能够增加微囊化益生菌在常温下储藏的稳定性,耐胃酸性能更好,肠溶效果优异。
(二)探究芯材组分含量对微囊化益生菌存活率的影响
1、为了探究不同水苏糖含量对微囊化益生菌存活率的影响,本发明进行了实施例17~20实验,实施例17~20实验的具体内容如下。
实施例17~实施例20:
实施例17~实施例20的内容与实施例1基本相同,不同之处在于:步骤(1)中菌悬液、水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液的质量比不同,具体参见表2。
称取实施例17~20制备的微囊化益生菌,进行常温下保存、模拟胃液中处理和模拟肠液中处理后取样进行活菌计数,然后根据活菌数计算结果,实验结果如表2所示。
由表2可以看出,在芯材混合液中益生菌:水苏糖溶液:乳果糖溶液:低聚异麦芽糖溶液:魔芋微粉溶液的质量比在1:1~10:2:2:3范围内,微囊化益生菌在常温保存60d后的存活率均在78.6%以上,在模拟胃液中微胶囊的抗酸能力较强,在模拟肠液中可以快速崩解,具有肠溶速度快,能够释放出大量的益生菌的特点。
2、为了探究乳果糖含量对微囊化益生菌存活率的影响,本发明进行了实施例21~23实验,实施例21~23实验的具体内容如下。
实施例21~23:
实施例21~实施例23的内容与实施例1基本相同,不同之处在于:步骤(1)中菌悬液、水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液的质量比不同,具体参见表3。
称取实施例21~23制备的微囊化益生菌,进行常温下保存、模拟胃液中处理和模拟肠液中处理后取样进行活菌计数,然后根据活菌数计算结果,实验结果如表3所示。
由表3可以看出,在芯材混合液中益生菌:水苏糖溶液:乳果糖溶液:低聚异麦芽糖溶液:魔芋微粉溶液的质量比在1:5:1~3:2:3范围内,微囊化益生菌在常温保存60d后的存活率均在79.8%以上,在模拟胃液和肠液中的存活率和释放率均较高。
3、为了探究低聚异麦芽糖含量对微囊化益生菌存活率的影响,本发明进行了实施例24~26实验,实施例24~26实验的具体内容如下。
实施例24~26:
实施例24~实施例26的内容与实施例1基本相同,不同之处在于:步骤(1)中菌悬液、水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液的质量比不同,具体参见表4。
称取实施例24~26制备的微囊化益生菌,进行常温下保存、模拟胃液中处理和模拟肠液中处理后取样进行活菌计数,然后根据活菌数计算结果,实验结果如表4所示。
由表4可以看出,在芯材混合液中益生菌:水苏糖溶液:乳果糖溶液:低聚异麦芽糖溶液:魔芋微粉溶液的质量比在1:5:2:1~3:3范围内,微囊化益生菌在常温保存60d后的存活率均在79.9 %以上,在模拟胃液和肠液中的存活率和释放率均较高。
4、为了探究魔芋微粉含量对微囊化益生菌存活率的影响,本发明进行了实施例27~29实验,实施例27~29实验的具体内容如下。
实施例27~29:
实施例27~实施例29的内容与实施例1基本相同,不同之处在于:步骤(1)中菌悬液、水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液的质量比不同,具体参见表5。
称取实施例27~29制备的微囊化益生菌,进行常温下保存、模拟胃液中处理和模拟肠液中处理后取样进行活菌计数,然后根据活菌数计算结果,实验结果如表5所示。
由表5可以看出,在芯材混合液中益生菌:水苏糖溶液:乳果糖溶液:低聚异麦芽糖溶液:魔芋微粉溶液的质量比在1:5:2:2:1~5范围内,微囊化益生菌在常温保存60d后的存活率均在79.2%以上,在模拟胃液和肠液中的存活率和释放率均较高。
实施例30:
(1)将两歧双歧杆菌、长双歧杆菌、鼠李糖乳杆菌于36℃条件下接种培养,发酵18h,得到益生菌发酵液。将益生菌发酵液在转速3000r/min离心10min,收集沉淀物,得到菌泥。将得到的菌泥用0.85%无菌生理盐水洗涤2~3次,将洗涤后的菌泥重悬于无菌水中,震荡混匀,得到每种菌浓度均为109 CFU/mL的菌悬液;
(2)将菌悬液、浓度为10%的水苏糖溶液、浓度为2%的乳果糖溶液、浓度为3%的低聚异麦芽糖溶液和浓度为0.1%的魔芋微粉溶液按质量比1:9:3:2:5混合,得到芯材混合液;
(3)将芯材混合液、浓度为8%的脱脂乳粉溶液和浓度为1%的海藻酸钠溶液按质量比1:15:18混合,得到混合液;
(4)将均匀挤压的混合液滴入浓度为3%的氯化钙溶液中,形成微囊,磁力搅拌器以转速300r/min搅拌20min,待微囊充分凝固后用蒸馏水冲洗3次,除去多余的钙离子和未被包埋的菌体,得到微囊化益生菌。
实施例31:
(1)将两歧双歧杆菌、长双歧杆菌、鼠李糖乳杆菌、罗伊氏乳杆菌、嗜酸乳杆菌于36℃条件下接种培养,发酵18h,得到益生菌发酵液。将益生菌发酵液在转速4000r/min离心10min,收集沉淀物,得到菌泥。将得到的菌泥用0.85%无菌生理盐水洗涤2~3次,将洗涤后的菌泥重悬于无菌水中,震荡混匀,得到每种菌浓度均为1011 CFU/mL的菌悬液;
(2)将菌悬液、浓度为12%的水苏糖溶液、浓度为4%的乳果糖溶液、浓度为5%的低聚异麦芽糖溶液和浓度为0.2%的魔芋微粉溶液按质量比1:7:2:2:4混合,得到芯材混合液;
(3)将芯材混合液、浓度为10%的脱脂乳粉溶液和浓度为2%的海藻酸钠溶液按质量比1:12:20混合,得到混合液;
(4)将均匀挤压的混合液滴入浓度为2%的氯化钙溶液中,形成微囊,磁力搅拌器以转速400r/min搅拌10min,待微囊充分凝固后用蒸馏水冲洗3次,除去多余的钙离子和未被包埋的菌体,得到微囊化益生菌。
实施例32:
(1)将长双歧杆菌、鼠李糖乳杆菌、罗伊氏乳杆菌、嗜酸乳杆菌于36℃条件下接种培养,发酵18h,得到益生菌发酵液。将益生菌发酵液在转速5000r/min离心5min,收集沉淀物,得到菌泥。将得到的菌泥用0.85%无菌生理盐水洗涤2~3次,将洗涤后的菌泥重悬于无菌水中,震荡混匀,得到每种菌浓度均为107 CFU/mL的菌悬液;
(2)将菌悬液、浓度为18%的水苏糖溶液、浓度为4%的乳果糖溶液、浓度为3%的低聚异麦芽糖溶液和浓度为0.6%的魔芋微粉溶液按质量比1:5:2:1:2混合,得到芯材混合液;
(3)将芯材混合液、浓度为8%的脱脂乳粉溶液和浓度为3%的海藻酸钠溶液按质量比1:12:8混合,得到混合液;
(4)将均匀挤压的混合液滴入浓度为2%的氯化钙溶液中,形成微囊,磁力搅拌器以转速500r/min搅拌10min,待微囊充分凝固后用蒸馏水冲洗3次,除去多余的钙离子和未被包埋的菌体,得到微囊化益生菌。
实施例33:
(1)将长双歧杆菌、鼠李糖乳杆菌、罗伊氏乳杆菌于36℃条件下接种培养,发酵18h,得到益生菌发酵液。将益生菌发酵液在转速6000r/min离心5min,收集沉淀物,得到菌泥。将得到的菌泥用0.85%无菌生理盐水洗涤2~3次,将洗涤后的菌泥重悬于无菌水中,震荡混匀,得到每种菌浓度均为109 CFU/mL的菌悬液;
(2)将菌悬液、浓度为20%的水苏糖溶液、浓度为3%的乳果糖溶液、浓度为5%的低聚异麦芽糖溶液和浓度为0.8%的魔芋微粉溶液按质量比1:3:1:1:2混合,得到芯材混合液;
(3)将芯材混合液、浓度为8%的脱脂乳粉溶液和浓度为2%的海藻酸钠溶液按质量比1:6:8混合,得到混合液;
(4)将均匀挤压的混合液滴入浓度为2%的氯化钙溶液中,形成微囊,磁力搅拌器以转速400r/min搅拌10min,待微囊充分凝固后用蒸馏水冲洗3次,除去多余的钙离子和未被包埋的菌体,得到微囊化益生菌。
上述实施例为本发明的具体实施方式,但本发明的实施方式并不受上述实施例的限制,其它任何不超出本发明设计思路组合、改变、修饰、替代、简化,均落入本发明的保护范围之内。
Claims (9)
1.一种含水苏糖和魔芋微粉的微囊化益生菌的制备方法,其特征在于,包括以下步骤:
(1)将菌悬液、水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液按质量比1:1~10:1~3:1~3:1~5混合,得到芯材混合液;
(2)将步骤(1)得到的芯材混合液与脱脂乳粉溶液和浓度为5%~20%的海藻酸钠溶液混合,得到混合液;
(3)将步骤(2)得到的混合液滴入氯化钙溶液中,形成微囊,微囊凝固后,过滤,除去水相,洗涤,得到微囊化益生菌。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述水苏糖溶液、乳果糖溶液、低聚异麦芽糖溶液和魔芋微粉溶液的浓度分别为10%~20%、2%~5%、2%~10%、0.1%~1%。
3.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述菌悬液中含有的益生菌为两歧双歧杆菌、长双歧杆菌、鼠李糖乳杆菌、罗伊氏乳杆菌、嗜酸乳杆菌中的至少一种。
4.根据权利要求3所述的制备方法,其特征在于,步骤(1)所述菌悬液中每种益生菌的浓度均为107~1011CFU/mL。
5.根据权利要求1所述的制备方法,其特征在于,步骤(2)中芯材混合液、脱脂乳粉溶液、海藻酸钠溶液的质量比为1:1~20:1~20。
6.根据权利要求1所述的制备方法,其特征在于,步骤(2)中脱脂乳粉溶液的浓度为5%~20%。
7.根据权利要求1所述的制备方法,其特征在于,步骤(3)中氯化钙溶液的浓度为1%~10%。
8.根据权利要求1所述的制备方法,其特征在于,步骤(3)形成微囊的过程中需要搅拌,搅拌速度为200~1000r/min,搅拌时间为10~30min。
9.一种利用权利要求1~8任一所述制备方法制备的含水苏糖和魔芋微粉的微囊化益生菌产品。
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