CN115104737A - 益生菌冻干干燥及包埋联动工艺 - Google Patents
益生菌冻干干燥及包埋联动工艺 Download PDFInfo
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Abstract
本发明公开了一种益生菌冻干干燥及包埋联动工艺,属于益生菌技术领域。所述益生菌冻干干燥的方法,主要包括以下步骤:培养基的制备、益生菌的培养、收集菌体、冷冻保护剂的制备、真空冷冻干燥及喷雾包埋。该方法通过冻干、制粒、喷雾包埋联动机制,提高了菌体在冷冻干燥时的存活率;通过制粒、喷雾包埋工艺良性运转,得到包埋物外观完整,加入成型的包衣芯材降低喷雾包埋工艺时间,益生菌包埋物在包埋工艺中损失较小;益生菌包埋物在室温储存期间保持了一定的生物活性;通过包埋工艺增强其储存中对抗外界环境温度的影响。
Description
技术领域
本发明属于益生菌技术领域,涉及一种益生菌冻干干燥及包埋联动工艺。
背景技术
冷冻干燥技术是将湿物料或溶液在较低的温度下冻结成固态,然后在真空下使其中的水分直接升华成气态,最终使物料脱水的干燥技术。冷冻干燥在低温下进行,因此对于许多热敏性的物质特别适用。如蛋白质、微生物之类通过冷冻干燥不会发生变性或失去生物活力。益生菌是通过定殖在人体内,改变宿主某一部位菌群组成的一类对宿主有益的活性微生物。益生菌的活性和长期稳定性可以受多个环境因素影响,例如,温度、酸碱度、液体环境(水)或者固体环境以及化学试剂等。当益生菌处于室温,处于液体环境(水)下中会很快失去活性,益生菌生命周期很短。因此,益生菌要长期保存,低温是比较行之有效的手段;或者使其处于固体状态,也可以延长益生菌生命周期。通常在制备保存周期较长的益生菌产品时,会将培育益生菌菌液在低温条件下进行固化。冷冻干燥是目前效果显著的益生菌低温固化技术,可以将含水量较大的益生菌菌泥干燥成益生菌菌粉,最大程度降低了干燥过程中益生菌的损耗。益生菌包埋是为了保证益生菌产品在储存期内的活性,通常经过包埋的益生菌产品较未经包埋的益生菌同类产品在同一的储存周期内,显示活性的指标菌数可以提高102。
中国专利申请CN 114292788 A公开了一种活性益生菌冷冻干燥的制备方法,包括以下步骤:培养基的制备、培养益生菌、收集菌体、配制保护剂以及真空冷冻干燥。该申请通过将可溶性淀粉、海藻糖、脱脂乳以及无机盐复合作为保护剂,通过四者的协同作用,提高了菌体在冷冻干燥时的存活率。该专利较好解决了在冻干干燥过程中益生菌菌数的快速下降问题,但未能解决益生菌室温储存问题及摄取过程中抗胃酸和胆碱问题。包埋是目前解决室温储存及使用中抗胃酸和胆碱问题一种有效的方法,将益生菌包埋在保护性结构中,能够增强其储存中对抗外界环境温度的影响及摄取过程,对抗胃酸和胆碱环境的影响,从而进一步提高益生菌的菌群活性,提高益生菌的实验效果。
因此,有必要探索一种处理过程中存活率高、可有效保持益生菌生物活性的冷冻干燥与包埋结合的工艺。
发明内容
本发明的目的是提供一种存活率高、损失量小、生物活性高的益生菌冻干干燥及包埋联动工艺。
为实现上述目的,本发明采用的技术方案如下:
首先,提供了一种益生菌冷冻干燥的方法,包括以下步骤:
(S1)培养基的制备:培养基以酵母蛋白胨、酵母浸粉、牛肉膏、氯化钠和纯化水为原料配制灭菌而成;
(S2)培养益生菌:将保藏的益生菌菌株接入所述培养基中进行培养,待培养至对数生长期时,将培养液冷却静置,得到益生菌发酵液;
(S3)收集菌体:将所述益生菌发酵液离心,弃去上清液,得到益生菌菌泥;
(S4)冷冻保护剂的制备:所述冷冻保护剂以海藻糖、麦芽糊精、维生素C钠、羟丙基甲基纤维素和纯化水为原料配制灭菌而成;
(S5)真空冷冻干燥:将所述益生菌菌泥与冷冻保护剂混合制得乳化液;所得乳化液进行真空冷冻干燥,制成冻干菌粉;
(S6)喷雾包埋:
(1)将所述冻干菌粉过筛,所得菌粉用酸处理淀粉、麦芽糊精、低聚果糖和纯化水进行喷雾包埋,干燥后得包埋物A;
(2)所得包埋物A用浓缩乳清蛋白、酪蛋白酸钠、低聚果糖、磷脂和纯化水进行喷雾包埋,干燥后得包埋物B;
(3)所得包埋物B用鲁普兰多糖、麦芽糊精、和纯化水进行喷雾包埋,干燥后得包埋益生菌颗粒。
进一步地,冷冻保护剂的制备中所述海藻糖、麦芽糊精、维生素C钠、羟丙基甲基纤维素和纯化水的重量比为6-10:10-11:1.0-1.4:0.1-0.5:20。
优选地,冷冻保护剂的制备中所述海藻糖、麦芽糊精、维生素C钠、羟丙基甲基纤维素和纯化水的重量比为7-9:10-11:1.0-1.4:0.2-0.4:20。
在冷冻保护剂中,海藻糖、麦芽糊精、维生素C钠能够降低益生菌在冷冻结晶过程中所受的伤害,羟丙基甲基纤维素作为一种水溶性粘合剂,可以调节冷冻保护剂稠度,使冻干物保持软硬适中的块状,便于下一步物理制粒。
进一步地,真空冷冻干燥中所述益生菌菌泥与冷冻保护剂的重量比为0.8-1.2:0.3-0.5。
进一步地,喷雾包埋步骤(1)中冻干菌粉过筛所用的网筛尺寸为55-65目,所述酸处理淀粉、麦芽糊精、低聚果糖和纯化水的重量比为7-9:3-5:1-3:100。
进一步地,喷雾包埋步骤(2)中所述浓缩乳清蛋白、酪蛋白酸钠、低聚果糖、磷脂和纯化水的重量比为3-5:1.5-2.5:0.9-1.1:0.05-0.15:20。
进一步地,喷雾包埋步骤(3)中所述鲁普兰多糖、麦芽糊精、和纯化水的重量比为8-12:7-9:100。
在喷雾包埋过程中所添加的辅料没有单独的功能作用,多种成分复合衣层起保护作用,对抗外界环境温度的影响及摄取过程,对抗胃酸和胆碱环境的影响。
进一步地,所述益生菌菌株为乳双歧杆菌、罗伊士乳杆菌、植物乳杆菌、长双歧杆菌、短双歧杆菌的任意一种。
进一步地,收集菌体中所述离心采用管道式离心,其中温度为4-10℃,转速为5000-14000r/min,时间为10-15min。
进一步地,所述真空冷冻干燥的步骤如下:将菌体保护剂悬浊液置于真空冷冻干燥机内,在温度为-40℃下预冻2-3h,再开启真空泵,抽真空至真空度为5-10Pa,真空冷冻干燥24-48h。
在一些具体的实施方式中,益生菌冷冻干燥的方法包括以下步骤:
(1)培养基的制备:将7.5g酵母蛋白胨、7.5g酵母浸粉、3g牛肉膏和0.3g氯化钠加入5L纯化水中,加热溶解,调节pH为6.5,配制好后,121℃灭菌20min,备用;
(2)培养益生菌:将保藏的25mL双歧杆菌菌种接种到上述的培养基中进行活化培养,培养基加上已灭菌的液体石蜡,37℃培养32h后形成活化培养液;取25mL活化培养液接种到上述的培养基中进行扩大培养,37℃培养32h后得到扩大培养液,将扩大培养液在10℃下,静置2h后得到发酵液;
(3)收集菌体:将上述发酵液在温度为4℃,转速为5000r/min的条件下管道式离心10min,弃去上清液,得到沉淀物,将沉淀物用等体积的无菌生理盐水在转速为3000r/min,离心7min弃去上清液得到益生菌菌泥;
(4)冷冻保护剂的制备:以海藻糖、麦芽糊精、维生素C钠、羟丙基甲基纤维素和纯化水为原料115℃下灭菌20min配制而成;
(5)真空冷冻干燥:将上述1kg益生菌菌泥与0.4kg保护剂搅拌均匀形成乳化液;将乳化液放置与真空干燥机内,在温度为-40℃下预冻2h后开启真空泵,抽真空至真空度为8Pa,真空冷冻干燥36h后形成冻干粉,冻干粉破碎后通过60目筛网,用80目筛网筛去细粉。60目菌粉喷雾包埋备用,过80目菌粉其他用途;
(6)喷雾包埋:
a)将60目菌粉作为芯材,用酸处理淀粉、麦芽糊精、低聚果糖和纯化水进行流化床喷雾包埋,干燥后得包埋物A;
b)将包埋物A用浓缩乳清蛋白、酪蛋白酸钠、低聚果糖、磷脂和纯化水进行流化床喷雾包埋,干燥后得包埋物B;
c)将包埋物B用鲁普兰多糖、麦芽糊精和纯化水进行流化床喷雾包埋,干燥后得包埋益生菌颗粒。
喷雾包埋的过程具体为:
流化床喷雾包埋进风口温度37-42℃,出风口温度32-37℃。由于芯材加入时已经具有一定的形状,可以明显缩短喷雾包埋工艺时间,缩短益生菌在超常温度中的时间,增加野生菌的生存。
相比于现有技术,本发明具有以下有益效果:
(1)通过冻干、制粒、喷雾包埋联动机制,提高了菌体在冷冻干燥时的存活率;
(2)通过制粒、喷雾包埋工艺良性运转,得到包埋物外观完整,加入成型的包衣芯材降低喷雾包埋工艺时间;益生菌包埋物在包埋工艺中损失较小;
(3)益生菌包埋物在室温储存期间保持了一定的生物活性;通过包埋工艺增强其储存中对抗外界环境温度的影响。
具体实施方式
值得说明的是,本发明中使用的原料均为普通市售产品,对其来源不做具体限定。
以下试剂来源,为示例性说明:本发明中使用的原料为食品或者食品添加剂,符合相应的食品标准或者食品添加剂标准GB2760。
实施例1
包埋益生菌颗粒的制备方法,具体包括以下步骤:
(1)培养基的制备:将7.5g酵母蛋白胨、7.5g酵母浸粉、3g牛肉膏和0.3g氯化钠加入5L纯化水中,加热溶解,调节pH为6.5,配制好后,121℃灭菌20min,备用;
(2)培养益生菌:将保藏的25mL双歧杆菌菌种接种到上述的培养基中进行活化培养,培养基加上已灭菌的液体石蜡,37℃培养32h后形成活化培养液;取25mL活化培养液接种到上述的培养基中进行扩大培养,37℃培养32h后得到扩大培养液,将扩大培养液在10℃下,静置2h后得到发酵液;
(3)收集菌体:将上述发酵液在温度为4℃,转速为5000r/min的条件下管道式离心10min,弃去上清液,得到沉淀物,将沉淀物用等体积的无菌生理盐水在转速为3000r/min,离心7min弃去上清液得到益生菌菌泥;
(4)冷冻保护剂的制备:以海藻糖8g、麦芽糊精10.5g、维生素C钠1.2g、羟丙基甲基纤维素0.3g和纯化水20g为原料115℃下灭菌20min配制而成;
(5)真空冷冻干燥:将上述1kg益生菌菌泥与0.4kg保护剂搅拌均匀形成乳化液;将乳化液放置与真空干燥机内,在温度为-40℃下预冻2h后开启真空泵,抽真空至真空度为8Pa,真空冷冻干燥36h后形成冻干粉,冻干粉破碎后通过60目筛网,用80目筛网筛去细粉。60目菌粉喷雾包埋备用,过80目菌粉其他用途。
(6)喷雾包埋:
a)将60目菌粉作为芯材,用酸处理淀粉8g、麦芽糊精4g、低聚果糖2g和纯化水100g进行流化床喷雾包埋,干燥后得包埋物A;
b)将包埋物A用浓缩乳清蛋白4g、酪蛋白酸钠2g、低聚果糖1g、磷脂0.1g和纯化水20g进行流化床喷雾包埋,干燥后得包埋物B;
c)将包埋物B用鲁普兰多糖10g、麦芽糊精8g和纯化水100g进行流化床喷雾包埋,干燥后得包埋益生菌颗粒;
流化床喷雾包埋进风口温度40℃,出风口温度35℃。
实施例2
与实施例1的不同在于步骤(4)中原料的用量为:海藻糖7g、麦芽糊精11g、维生素C钠1.0g、羟丙基甲基纤维素0.4g、纯化水20g,其他步骤同实施例1。
实施例3
与实施例1的不同在于步骤(4)中原料的用量为:海藻糖9g、麦芽糊精10g、维生素C钠1.4g、羟丙基甲基纤维素0.2g、纯化水20g,其他步骤同实施例1。
实施例4
与实施例1的不同在于步骤(4)中原料的用量为:海藻糖6g、麦芽糊精11g、维生素C钠1.0g、羟丙基甲基纤维素0.5g、纯化水20g,其他步骤同实施例1。
实施例5
与实施例1的不同在于步骤(4)中原料的用量为:海藻糖10g、麦芽糊精10g、维生素C钠1.4g、羟丙基甲基纤维素0.1g、纯化水20g,其他步骤同实施例1。
实施例6
与实施例1的不同在于步骤(6)中原料的用量为:a)用酸处理淀粉7g、麦芽糊精5g、低聚果糖1g、纯化水100g,其他步骤同实施例1。
实施例7
与实施例1的不同在于步骤(6)中原料的用量为:a)用酸处理淀粉9g、麦芽糊精3g、低聚果糖3g、纯化水100g,其他步骤同实施例1。
实施例8
与实施例1的不同在于步骤(6)中原料的用量为:b)浓缩乳清蛋白3g、酪蛋白酸钠2.5g、低聚果糖0.9g、磷脂0.12g、纯化水20g,c)鲁普兰多糖8g、麦芽糊精9g、纯化水100g,其他步骤同实施例1。
实施例9
与实施例1的不同在于步骤(6)中原料的用量为:b)浓缩乳清蛋白5g、酪蛋白酸钠1.5g、低聚果糖1.1g、磷脂0.07g、纯化水20g,c)鲁普兰多糖12g、麦芽糊精7g、纯化水100g,其他步骤同实施例1。
实施例10
与实施例1的不同在于步骤(4)中原料的用量为:海藻糖11g、麦芽糊精8g、维生素C钠0.9g、羟丙基甲基纤维素0.6g、纯化水20g,其他步骤同实施例1。
实施例11
与实施例1的不同在于步骤(6)中原料的用量为:a)用酸处理淀粉9.5g、麦芽糊精2g、低聚果糖3.5g、纯化水100g,其他步骤同实施例1。
实施例12
与实施例1的不同在于步骤(6)中原料的用量为:b)浓缩乳清蛋白2.5g、酪蛋白酸钠3.5g、低聚果糖0.85g、磷脂0.15g、纯化水20g,其他步骤同实施例1。
实施例13
与实施例1的不同在于步骤(6)中原料的用量为:c)鲁普兰多糖12.5g、麦芽糊精6.5g、纯化水100g,其他步骤同实施例1。
对比例1
使用常规工艺,不进行包埋处理,即与实施例1相比缺少步骤(6)。
实验例
采用相同重量的由实施例1-13获得的包埋益生菌颗粒进行性能测试,结果如表1。对比例1没有设置成完整的包埋益生菌颗粒的制备工艺,即缺少包埋步骤,因此不对其进行存活率的性能测试。
存活率的测定:将冻干菌粉包埋后,加入灭菌的生理盐水复水30min,用微量移液器吸取与冻干前等量的1mL菌液进行稀释涂板计数。测定冻干前、包埋后的活菌数目,最终计算出冷冻存活率,存活率(%)=样品包埋后的活菌总数/样品冻干前的活菌总数×100%。
表1 性能检测数据表
编号 | 存活率(%) |
实施例1 | 57.8 |
实施例2 | 56.2 |
实施例3 | 55.0 |
实施例4 | 55.2 |
实施例5 | 54.5 |
实施例6 | 54.2 |
实施例7 | 55.3 |
实施例8 | 54.5 |
实施例9 | 56.3 |
实施例10 | 53 |
实施例11 | 53.5 |
实施例12 | 52.9 |
实施例13 | 53.3 |
以乳双歧杆菌为例,对实施例1-13、对比例1进行稳定性试验测试,观察0月、6月、12月储存期内测试样品菌数(单位:CFU/g),储存条件:样品铝箔密封,测试结果见表2。
表2 稳定性测试(温度4℃)
0月 | 6月 | 12月 | |
实施例1 | 1.5*10<sup>11</sup> | 5.0*10<sup>10</sup> | 8.0*10<sup>9</sup> |
实施例2 | 1.6*10<sup>11</sup> | 5.2*10<sup>10</sup> | 8.1*10<sup>9</sup> |
实施例3 | 1.6*10<sup>11</sup> | 5.3*10<sup>10</sup> | 8.3*10<sup>9</sup> |
实施例4 | 1.5*10<sup>11</sup> | 4.8*10<sup>10</sup> | 7.5*10<sup>9</sup> |
实施例5 | 1.6*10<sup>11</sup> | 5.1*10<sup>10</sup> | 8.0*10<sup>9</sup> |
实施例6 | 1.4*10<sup>11</sup> | 4.4*10<sup>10</sup> | 6.9*10<sup>9</sup> |
实施例7 | 1.5*10<sup>11</sup> | 4.9*10<sup>10</sup> | 7.7*10<sup>9</sup> |
实施例8 | 1.5*10<sup>11</sup> | 4.9*10<sup>10</sup> | 7.6*10<sup>9</sup> |
实施例9 | 1.4*10<sup>11</sup> | 4.5*10<sup>10</sup> | 7.0*10<sup>9</sup> |
实施例10 | 1.5*10<sup>11</sup> | 4.5*10<sup>10</sup> | 6.8*10<sup>9</sup> |
实施例11 | 1.4*10<sup>11</sup> | 4.2*10<sup>10</sup> | 6.4*10<sup>9</sup> |
实施例12 | 1.5*10<sup>11</sup> | 4.4*10<sup>10</sup> | 6.7*10<sup>9</sup> |
实施例13 | 1.6*10<sup>11</sup> | 4.7*10<sup>10</sup> | 7.2*10<sup>9</sup> |
对比例1 | 2.1*10<sup>11</sup> | 2.0*10<sup>8</sup> | 3.0*10<sup>7</sup> |
根据稳定性测试结果,包埋的菌粉对比未包埋的菌粉,6月、12月测试的菌数结果高两个数量级,也就是说包埋菌粉稳定性较好。
最后应当说明的是,以上内容仅用以说明本发明的技术方案,而非对本发明保护范围的限制,本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换,均不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种益生菌冷冻干燥的方法,其特征在于,包括以下步骤:
(S1)培养基的制备:培养基以酵母蛋白胨、酵母浸粉、牛肉膏、氯化钠和纯化水为原料配制灭菌而成;
(S2)培养益生菌:将保藏的益生菌菌株接入所述培养基中进行培养,待培养至对数生长期时,将培养液冷却静置,得到益生菌发酵液;
(S3)收集菌体:将所述益生菌发酵液离心,弃去上清液,得到益生菌菌泥;
(S4)冷冻保护剂的制备:所述冷冻保护剂以海藻糖、麦芽糊精、维生素C钠、羟丙基甲基纤维素和纯化水为原料配制灭菌而成;
(S5)真空冷冻干燥:将所述益生菌菌泥与冷冻保护剂混合制得乳化液;所得乳化液进行真空冷冻干燥,制成冻干菌粉;
(S6)喷雾包埋:
(1)将所述冻干菌粉过筛,所得菌粉用酸处理淀粉、麦芽糊精、低聚果糖和纯化水进行喷雾包埋,干燥后得包埋物A;
(2)所得包埋物A用浓缩乳清蛋白、酪蛋白酸钠、低聚果糖、磷脂和纯化水进行喷雾包埋,干燥后得包埋物B;
(3)所得包埋物B用鲁普兰多糖、麦芽糊精、和纯化水进行喷雾包埋,干燥后得包埋益生菌颗粒。
2.根据权利要求1所述的方法,其特征在于,冷冻保护剂的制备中所述海藻糖、麦芽糊精、维生素C钠、羟丙基甲基纤维素和纯化水的重量比为6-10:10-11:1.0-1.4:0.1-0.5:20。
3.根据权利要求2所述的方法,其特征在于,冷冻保护剂的制备中所述海藻糖、麦芽糊精、维生素C钠、羟丙基甲基纤维素和纯化水的重量比为7-9:10-11:1.0-1.4:0.2-0.4:20。
4.根据权利要求1所述的方法,其特征在于,真空冷冻干燥中所述益生菌菌泥与冷冻保护剂的重量比为0.8-1.2:0.3-0.5。
5.根据权利要求1所述的方法,其特征在于,喷雾包埋步骤(1)中冻干菌粉过筛所用的网筛尺寸为55-65目,所述酸处理淀粉、麦芽糊精、低聚果糖和纯化水的重量比为7-9:3-5:1-3:100。
6.根据权利要求1所述的方法,其特征在于,喷雾包埋步骤(2)中所述浓缩乳清蛋白、酪蛋白酸钠、低聚果糖、磷脂和纯化水的重量比为3-5:1.5-2.5:0.9-1.1:0.07-0.12:20。
7.根据权利要求1所述的方法,其特征在于,喷雾包埋步骤(3)中所述鲁普兰多糖、麦芽糊精和纯化水的重量比为8-12:7-9:100。
8.根据权利要求1所述的方法,其特征在于,所述益生菌菌株为乳双歧杆菌、罗伊士乳杆菌、植物乳杆菌、长双歧杆菌、短双歧杆菌的任意一种。
9.根据权利要求1所述的方法,其特征在于,收集菌体中所述离心采用管道式离心,其中温度为4-10℃,转速为5000-14000r/min,时间为10-15min。
10.根据权利要求1所述的方法,其特征在于,所述真空冷冻干燥的步骤如下:将菌体保护剂悬浊液置于真空冷冻干燥机内,在温度为-40℃下预冻2-3h,再开启真空泵,抽真空至真空度为5-10Pa,真空冷冻干燥24-48h。
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