CN109566893A - 一种应用于畜牧养殖的益生菌添加剂的制备方法 - Google Patents
一种应用于畜牧养殖的益生菌添加剂的制备方法 Download PDFInfo
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- CN109566893A CN109566893A CN201910097428.5A CN201910097428A CN109566893A CN 109566893 A CN109566893 A CN 109566893A CN 201910097428 A CN201910097428 A CN 201910097428A CN 109566893 A CN109566893 A CN 109566893A
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Classifications
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- A—HUMAN NECESSITIES
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Abstract
本发明涉及畜牧养殖饲料添加剂领域,尤其涉及一种应用于畜牧养殖的益生菌添加剂的制备方法;包括将益生菌发酵上清液进行调节pH值、添加稳定剂、添加抗氧化剂、添加益生元、以及杀菌处理的步骤;其中,所述益生菌发酵上清液为包括益生菌的发酵上清和菌体在内的整个发酵后处理溶液;本发明的应用于畜牧养殖的益生菌添加剂的制备方法,贮存稳定、应用于饲料中对家猪的生长性能有明显促进作用,既环保又经济节约。
Description
技术领域
本发明涉及畜牧养殖饲料添加剂领域,尤其涉及一种应用于畜牧养殖的益生菌添加剂的制备方法。
背景技术
益生菌作为一种肠道微生物,对宿主具有调节肠道菌群平衡,抑制有害菌,提高宿主免疫力,促进消化吸收等作用。其作用机理主要是其生长过程中所产生的代谢产物,如乳酸、细菌素、胞外多糖等,对宿主产生的有益调节作用。
益生菌企业的发酵产品,多数通过对菌株进行种子扩培、发酵、离心收集菌体后进行冻干或喷雾干燥得到菌粉,其中离心后的上清液,通常是直接作为废水,经过处理后排掉,处理过程不仅繁琐,还需要企业为此需要支付一定的处理费用。
然而,发酵上清液通常含有少量活的菌体、多种营养物质、菌体的代谢产物(乳酸、细菌素、胞外多糖等)等,若能解决长期放置产生杂菌、变质的问题,将其有效利用,则既环保,又经济节约。
发明内容
为解决上述技术问题,本发明的目的是提供一种贮存稳定、应用于饲料中对家猪的生长性能有明显促进作用,既环保又经济节约的应用于畜牧养殖的益生菌添加剂的制备方法。
本发明的应用于畜牧养殖的益生菌添加剂的制备方法,包括将益生菌发酵上清液进行调节pH值、添加抗氧化剂、添加益生元、以及杀菌处理的步骤。
进一步的,将益生菌发酵上清液调节pH值至2.5-3.5。
进一步的,用于调节pH值的酸性溶液为乳酸、柠檬酸、盐酸、硝酸中的一种或多种。
进一步的,所述抗氧化剂包括a-硫辛酸、D-异抗坏血酸、D-异抗坏血酸钠、抗坏血酸钠、抗坏血酸钙中的一种或多种。
进一步的,所述抗氧化剂包括浓度为0.01-0.1g/L的a-硫辛酸、0.2-2.0g/L的D-异抗坏血酸、0.2-1.0g/L的D-异抗坏血酸钠、0.2-1.0g/L的抗坏血酸钠、0.1-1.0g/L的抗坏血酸钙中的一种或多种。
进一步的,所述益生元包括低聚果糖、低聚木糖、低聚半乳糖、聚葡萄糖、低聚异麦芽糖、水苏糖、菊粉中的一种或多种。
进一步的,所述益生元包括浓度为1-10g/L的低聚果糖、1-10g/L的低聚木糖、0.5-5g/L的低聚半乳糖、1-10g/L的聚葡萄糖、1-10g/L的低聚异麦芽糖、0.5-5g/L的水苏糖、1-10g/L的菊粉中的一种或多种。
进一步的,所述杀菌处理的条件为:巴氏杀菌法,60-70℃处理30min。
具体的,所述益生菌包括乳杆菌属和双歧杆菌属益生菌。
具体的,所述乳杆菌属益生菌包括嗜酸乳杆菌、干酪乳杆菌、卷曲乳杆菌、德氏乳杆菌保加利亚亚种、德氏乳杆菌乳亚种、发酵乳杆菌、格氏乳杆菌、瑞士乳杆菌、约氏乳杆菌、副干酪乳杆菌、植物乳杆菌、罗伊氏乳杆菌、鼠李糖乳杆菌、唾液乳杆菌、凝结芽孢杆菌、清酒乳杆菌、产丙酸丙酸杆菌。
具体的,所述双歧杆菌属益生菌包括青春双歧杆菌、乳双歧杆菌、两歧双歧杆菌、短双歧杆菌、婴儿双歧杆菌、长双歧杆菌。
具体的,所述益生菌发酵上清液的制备方法为:所述益生菌采用液体培养基,控制一定的发酵条件,发酵培养结束后,离心收集上清液,得到益生菌发酵上清液;离心条件为管式离心机8000-10000rpm。
具体的,所述液体培养基包括5-20g/L的蛋白胨、5-15g/L的酵母浸粉、10-40g/L的葡萄糖、1-10g/L的无水乙酸钠、1-5g/L的柠檬酸氢二铵、0.5-1.5g/L的吐温-80、1-5g/L的磷酸氢二钾、0.5-0.9g/L的硫酸锰、0.2-0.5g/L的硫酸镁。
具体的,所述发酵调节为:温度30-37℃,转速20-50rpm,初始pH控制6.0-7.0,采用氢氧化钠或碳酸钠维持pH在5.0-6.0。
借由上述方案,本发明至少具有以下优点:
1)本发明通过特定的温度对发酵上清液进行杀菌处理,杀死上清液中的一些活的菌体,同时降低因高温对上清液中的一些活性代谢产物的影响;
2)益生菌发酵上清液,通过调节pH值、添加一定的抗氧化剂,防止发酵上清液因贮存过程中微生物的生长而发生变质的情况出现,延长其贮存期;
3)益生菌发酵上清液中添加一定的益生元成份,应用于畜牧养殖的过程中,能调节宿主的肠道菌群平衡,增加肠道中有益微生物的数量,降低有害菌,提高饲料的利用率,降低料肉比,提高养殖效率;
4)本发明制备的发酵上清液理化性质稳定;应用于畜牧养殖中,能提升饲料的利用率,提高养殖报酬;同时也解决益生菌生产企业上清液废水处理的问题,推进企业的可持续发展。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例详细说明如后。
具体实施方式
下面结合实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
应当说明的是,下述实施例中的实验方法,如无特殊说明,均为常规方法;实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
由于益生菌发酵过程并非本发明的重点,本发明采用的常见试剂的配方如下,在后续实施例中不再赘述。
MRS培养基:将蛋白胨10.00g、牛肉膏10.00g、酵母膏5.00g、柠檬酸氢二铵2.00g、葡萄糖20.00g、吐温-80 1ml、磷酸氢二钾2.00g、硫酸锰0.58g和硫酸镁0.28g溶于1L蒸馏水,调节pH值至6.4。
发酵培养基:5-20g/L蛋白胨、5-15g/L酵母浸粉、10-40g/L葡萄糖、1-10g/L无水乙酸钠、1-5g/L柠檬酸氢二铵、0.5-1.5g/L吐温-80、1-5g/L磷酸氢二钾、0.5-0.9g/L硫酸锰、0.2-0.5g/L硫酸镁。
实验例1发酵上清液的收集
虽然各益生菌的发酵方法不同,但是后续用于畜牧养殖的发酵上清液的处理方式差异不大,因而,本实施例仅以植物乳杆菌为例,做进一步说明,并提供一种益生菌发酵以及发酵上清液收集的方法,具体步骤如下:
S1、取植物乳杆菌菌株甘油管,接种至MRS培养基(装液量9ml的试管)中,35-37℃,厌氧静置培养18-24h,命名为体系1。
S2、取体系1,以1-2%的接种量接种至MRS培养基(装液量200-400ml)中,35-37℃,厌氧静置培养14-18h,命名为体系2。
S3、取体系2,以1-2%的接种量接种至MRS培养基(装液量10-20L)中,35-37℃,厌氧静置培养8-12h,命名为体系3。
S4、取体系3,以1-2%的接种量接种至发酵培养基(装液量500-1000L)中进行培养,初始pH控制6.0-7.0,采用氢氧化钠或碳酸钠维持pH在5.0-6.0,培养时间9-15h,命名为体系4。
S5、取体系4,采用管式离心机进行离心,转速8000-10000转/分,流速1000-2500L/h,上清液OD600值控制在0.5以下,收集发酵上清液。
实验例2发酵上清液的处理
本实施例采用实施例1获得的发酵上清液,进一步提供一种发酵上清液的处理、并最终制备为益生菌添加剂的对比试验方法,具体如下:
1、pH值的选择
本领域的研发人员应当了解,微生物在较低pH值条件下不容易生长,然而,对于不同的溶液而言,由于成分不同,其抑制微生物生长的最佳pH值并不一样。为此,本实施例将发酵上清液在不同pH值的条件下,常温下放置一定时间,检测溶液中的霉母酵母菌、细菌总数,在不同pH值下,达到霉母酵母菌为50CFU/ml(计数方法参照GB 478915-2010,下同)、细菌总数为1×103CFU/ml(计数方法参照GB 47892-2010,下同)所需要的天数,结果如表1所示:
表1不同pH值下霉母酵母菌、细菌总数变化状况
根据表1结果可知,pH值为3.0-3.5和2.5-3.0时,其放置一定时间后,抑菌效果较好,且其细菌总数达到1×103CFU/ml所需要的时间差异不大,因此后续实施例中相应步骤采用pH值为2.5-3.5。
2、抗氧化剂选择
本领域的研发人员应当了解,微生物在添加有抗氧化剂的溶液中不容易生长,然而,对于不同的溶液而言,由于成分不同,添加不同抗氧化剂的效果并不相同。为此,本实施例添加相同量的不同种类的抗氧化剂,在pH值为4.5的条件下,常温下放置一定时间后,检测溶液中的霉母酵母菌、细菌总数,达到霉母酵母菌为50CFU/ml、细菌总数为1×103CFU/ml所需要的天数,结果如表2所示:
表2添加不同抗氧化剂条件下霉母酵母菌、细菌总数变化状况
根据表2结果可知,添加a-硫辛酸抗氧化剂,其存储时间均优于其它抗氧化剂,因此后续实施例中抗氧化剂采用a-硫辛酸进行后续试验。
针对a-硫辛酸的用量,设置不同的用量梯度考察其最佳添加量,在pH值为4.5的条件下,常温下放置一定时间后,检测溶液中的霉母酵母菌、细菌总数,达到霉母酵母菌为50CFU/ml、细菌总数为1×103CFU/ml所需要的天数,结果如表3所示:
表3添加不同浓度a-硫辛酸下霉母酵母菌、细菌总数变化状况
根据表3结果可知,添加不同浓度的a-硫辛酸抗氧化剂,其添加为0.01-0.1g/L时,其贮存时间较好,继续添加其a-硫辛酸的用量,对贮存时间影响不大,因此,选择0.01-0.1g/L浓度的a-硫辛酸作为最佳添加量。
其它种类的抗氧化剂,如D-异抗坏血酸、D-异抗坏血酸钠、抗坏血酸钠、抗坏血酸钙,虽然效果没有a-硫辛酸好,但是仍然对其最适宜浓度做了如上的对比试验,结果表明,浓度为0.2-2.0g/L的D-异抗坏血酸、0.2-1.0g/L的D-异抗坏血酸钠、0.2-1.0g/L的抗坏血酸钠、或0.1-1.0g/L的抗坏血酸钙,抑菌效果较好。
3、热处理时间选择
为了进一步延长其发酵上清液的储存时间,本实施例还对发酵上清液进行热处理杀菌,考察不同温度对其上清液的抑菌能力、储存时间的影响。具体方式包括,在常温下放置一定时间后,其液体对大肠杆菌的抑菌能力(用抑菌圈表示),以及霉母酵母菌达到50CFU/ml、细菌总数达到1×103CFU/ml所需要的天数如表4所示:
表4发酵上清液不同热处理灭菌条件下抑菌效果对比表
其中,表4中“++”表示对大肠杆菌的抑菌圈直径能达到11-14mm;+”表示对大肠杆菌的抑菌圈直径能达到5-11mm;
根据表4结果可知,经过加热处理后,发酵上清液的储存时间明显延长,但考虑到温度过高对发酵上清液的抑菌致病菌的能力产生不利影响,以及考虑大规模生产的能耗问题,最佳的加热杀菌条件为,60℃处理30min。
4、最优处理方案
结合以上试验结果,本实施例提供一种益生菌添加剂的制备方法,具体步骤如下:
S1、采用柠檬酸,将益生菌发酵上清液调节pH值至2.5-3.5。
S3、向发酵上清液中加入0.01-0.1g/L的a-硫辛酸抗氧化剂,然后搅拌均匀。
S4、向发酵上清液中加入益生元,包括浓度为1-10g/L的低聚果糖、1-10g/L的低聚木糖、0.5-5g/L的低聚半乳糖、1-10g/L的聚葡萄糖、1-10g/L的低聚异麦芽糖、0.5-5g/L的水苏糖、1-10g/L的菊粉中的一种或多种。
S5、巴氏杀菌60-70℃处理30min。
将处理完的发酵上清液,常温下放置一定时间后,检测溶液中的霉母酵母菌、细菌总数,达到霉母酵母菌为50CFU/ml、细菌总数为1×103CFU/ml所需要的天数,结果如表5所示:
表5最佳处理方案下发酵上清液的抑菌效果表
其中,表5中“-”表示在一定时间内,其霉菌酵母菌的数量没有达到50CFU/ml或细菌总数没有达到1×103CFU/ml;“+”表示在一定时间内,其霉菌酵母菌的数量达到50CFU/ml或细菌总数达到1×103CFU/ml;
由以上数据显示,采用最终最佳的条件,最终使储存时间延长至3个月。满足其上清液应用于畜牧养殖的需求。
实验例3益生菌发酵上清液的应用
一、试验动物
试验动物:按照遗传背景相同、体重相近、窝别相同的原则,选取体重约16-18kg的“杜×长×大”三元杂交猪。
二、试验材料
1、益生菌发酵上清液
选用通过实施例1和2方法制备的益生菌发酵上清液。
2、试验日粮
日粮配方以质量分数计包括:玉米55.24%、大麦7%、膨化大豆6.23%、脱皮豆粕20.68%、次粉6%、棉粕4%、豆油0.5%、食盐0.35%、以及维生素和微量元素。
对照组日粮中,日粮与水以1:2~4的比例进行混合、饲喂;试验组日粮中,日粮与益生菌发酵上清液同样以1:2~4的比例进行混合、饲喂。
三、试验方法
1、动物饲养试验
选取体重约16-18kg的“杜×长×大”三元杂交猪60头,随机分为2组,每组3个重复,每个重复10头。对照组、试验组按本配方配制日粮饲喂。试验按照猪的生长阶段分为前期(20-40kg)、中期(40-80kg)和后期(80-120kg)3个阶段。
试验期内提供适宜的温度湿度,地面饲养,通风,自由采食,饮水。免疫消毒程序按照猪场常规方法进行。
在实验开始、第26天(20-40kg)、第72天(40-80kg)及实验结束(80-120kg)前1天20:00断料供水,次日08:00以重复为单位对所有试验猪进行空腹称体重,结算相应试验期的耗料情况,计算平均增重、日采食量和料重比。
2、粪便中大肠杆菌菌群的测定
在试验的不同阶段,每组随机采食10头育肥猪的新鲜粪样,用无菌塑料袋封装,采用平板涂布法进行细菌计数。
3、数据统计分析
试验数据采用SPSS 20.0统计分析软件处理数据,采用单因素方差分析One wayANOVA进行显著性检验,并采用Duncan氏法进行多重比较,以p<0.05作为差异显著判断标准,试验结果以“平均数±标准差”表示。
4、结果与分析
(1)益生菌上清液对生长肥育猪生长性能的影响
结果如表6所示,经统计学分析,试验组与对照组之间的末重、平均日增重、以及料重比的差异具有显著性(p value<0.05),即说明益生菌发酵上清液对生长肥育猪生长性能有一定促进作用,降低料重比,提高饲料转化率。
表6益生菌上清液对生长肥育猪生长性能的影响(n=4)
组别 | 初始重/kg | 末重/kg | 平均日增重/kg | 平均日采食量/kg | 料重比 |
对照组 | 16.45±0.74 | 97.3±1.29 | 0.58±0.08 | 0.98±0.05 | 1.86±0.05 |
试验组 | 16.40±0.63 | 111.51±1.16 | 0.69±0.06 | 1.10±0.04 | 1.74±0.14 |
(2)益生菌上清液对生长肥育猪粪便中大肠杆菌菌群的影响
结果如表7所示。通过对其试验的三个不同生长时期(20-40kg、40-80kg、80-120kg)的重量进行检测肥育猪中粪便中大肠杆菌的数量,显示试验组中的大肠杆菌数量均低于对照组(p<0.05),说明采用本发明的益生菌上清液能显著降低肥育猪肠道中的大肠杆菌数量。
表7益生菌上清液对生长肥育猪粪便中大肠杆菌菌群的影响(lg(CFU/g))
以上所述仅是本发明的优选实施方式,并不用于限制本发明,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。
Claims (10)
1.一种应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:包括将益生菌发酵上清液进行调节pH值、添加抗氧化剂、添加益生元、以及杀菌处理的步骤。
2.根据权利要求1所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:将益生菌发酵上清液调节pH值至2.5-3.5。
3.根据权利要求1所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:用于调节pH值的酸性溶液为乳酸、柠檬酸、盐酸、硝酸中的一种或多种。
4.根据权利要求1所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:所述抗氧化剂包括a-硫辛酸、D-异抗坏血酸、D-异抗坏血酸钠、抗坏血酸钠、抗坏血酸钙中的一种或多种。
5.根据权利要求4所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:所述抗氧化剂包括浓度为0.01-0.1g/L的a-硫辛酸、0.2-2.0g/L的D-异抗坏血酸、0.2-1.0g/L的D-异抗坏血酸钠、0.2-1.0g/L的抗坏血酸钠、0.1-1.0g/L的抗坏血酸钙中的一种或多种。
6.根据权利要求1所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:所述杀菌处理的条件为:巴氏杀菌法,60-70℃处理30min。
7.根据权利要求1所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:所述益生元包括低聚果糖、低聚木糖、低聚半乳糖、聚葡萄糖、低聚异麦芽糖、水苏糖、菊粉中的一种或多种。
8.根据权利要求7所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:所述益生元包括浓度为1-10g/L的低聚果糖、1-10g/L的低聚木糖、0.5-5g/L的低聚半乳糖、1-10g/L的聚葡萄糖、1-10g/L的低聚异麦芽糖、0.5-5g/L的水苏糖、1-10g/L的菊粉中的一种或多种。
9.根据权利要求1所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:所述益生菌发酵上清液的制备方法为:所述益生菌采用液体培养基,控制一定的发酵条件,发酵培养结束后,离心收集上清液,得到益生菌发酵上清液;离心条件为管式离心机8000-10000rpm。
10.根据权利要求9所述的应用于畜牧养殖的益生菌添加剂的制备方法,其特征在于:所述发酵调节为:温度30-37℃,转速20-50rpm,初始pH控制6.0-7.0,采用氢氧化钠或碳酸钠维持pH在5.0-6.0。
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