CN114521608B - 一种乳酸菌和芽孢杆菌的发酵增效组合物 - Google Patents
一种乳酸菌和芽孢杆菌的发酵增效组合物 Download PDFInfo
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- CN114521608B CN114521608B CN202210111916.9A CN202210111916A CN114521608B CN 114521608 B CN114521608 B CN 114521608B CN 202210111916 A CN202210111916 A CN 202210111916A CN 114521608 B CN114521608 B CN 114521608B
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- fermentation
- bacillus
- synergistic composition
- luteolin
- lactic acid
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Classifications
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Abstract
本发明提供了一种乳酸菌和芽孢杆菌的发酵增效组合物,属于微生物发酵领域。本发明所述的发酵增效组合物包括谷氨酸、苏氨酸、木犀草素、丹参素、硫酸锌、硫酸镁和D‑甘露糖醇。本发明首次发现将木犀草素和丹参素按照比例添加到增效组合物中,进一步应用于微生物发酵饲料制备中,可以提高微生物发酵饲料中乳酸菌和芽孢杆菌的活菌数同时提高发酵后产品中乳酸菌和芽孢杆菌的保存时限,具有良好的应用前景。
Description
技术领域
本发明涉及微生物发酵领域,具体涉及一种乳酸菌和芽孢杆菌的发酵增效组合物。
背景技术
由于耐药性问题,抗生素在饲料中的应用受到严格限制。而益生菌是一类通过调控动物肠道微生物区系的平衡,从而有效促进宿主动物健康和生长的微生物活菌制剂,且对动物和人无毒副作用。目前常用的益生菌主要有乳酸菌、芽孢杆菌等。
芽孢杆菌,属于芽孢杆菌科、芽孢杆菌属,是一类能产生抗力内生孢子的革兰氏阳性菌,细胞呈杆状且外层覆盖大量的吡啶二羧酸钙。其皮层位于核心和芽孢壳之间,含丰富的肽聚糖;核心是一种高度浓缩的、惰性的染色体;最外层的外壁为一层肽聚糖壁,且一层或多层的成分为蛋白质的芽孢衣。由于芽孢具有厚而含水量低的多层结构,所以折光性强,对染料不易着色,稳定性好,耐氧化、耐挤压、耐高温,能长期耐受60℃高温,在温度120℃下能存活20min,且耐酸碱,在胃酸环境中能保持活性,这可能与芽孢独具的高含量吡啶二羧酸有关。同时还具有广谱杆菌活性,能产生细菌素,抑制病原菌。目前应用较多的有枯草芽孢杆菌、地衣芽孢杆菌、蜡样芽孢杆菌等类型。芽孢杆菌耐受动物胃液,在动物肠道中生长萌发,且分泌多种酶类,促进动物体消化吸收,同时在肠道内竞争性夺氧,形成厌氧环境,有利于乳酸菌、双歧杆菌等厌氧菌的生长,抑制需氧肠杆菌的繁殖,从而减少消化道疾病的发生减少。
乳酸菌是指一类发酵糖类产乳酸的革兰氏阳性无芽孢的细菌,它不是一个严格意义上的微生物分类单位,凡是能从葡萄糖或乳糖的发酵过程中产生乳酸的细菌统称为乳酸菌。其包含许多种属,大多数乳酸菌不运动,少数以周毛运动,菌体常排列成链。乳酸链球菌族,菌体球状,通常成对或成链。乳酸杆菌族,菌体杆状,单个或成链,有时成丝状,产生假分枝。普通的乳酸菌,活力极弱,只能在相对受限制的环境中存活,一但脱离这些环境,其自身就会遭到灭亡。乳酸菌作为益生菌是最早被批准用于饲料添加剂的微生物,同时也是批准使用最多的一类微生物,主要作为微生态制剂应用的有乳酸杆菌、肠球菌、双歧杆菌。
微生物发酵饲料主要是以微生物发酵生产动物饲料的技术,不但能将禽畜难以消化吸收的粗蛋白、淀粉等大分子物质分解转化为易消化吸收的葡萄糖、氨基酸和维生素等小分子营养物质,而且能降解粗纤维,产生大量的生物活性物质,从而提高饲料的消化吸收率和营养价值。另外,微生物发酵饲料中的有益微生物还能杀死病原菌,维持动物体内微生态平衡,增强机能的免疫力,具有一定的防病治病作用。然而,现有微生物发酵饲料通常会存在杂菌多,有益菌活菌数低,发酵周期长等问题,大大降低了微生物发酵饲料的作用。专利CN201410355239.0提供了一种酵母菌、乳酸菌、芽孢杆菌固体发酵的增效剂,包括:赖氨酸0.2-3g/L、山梨糖醇100-500g/L、海藻糖600-1000g/L、硫酸钴0.023-0.12g/L、硫酸锌0.1-0.45g/L、酵母硒0.1-0.5g/L、甘露聚糖酶100000-200000U/L,不仅可以增加酵母菌、芽孢杆菌或乳酸菌单菌/混菌发酵后的益生菌的活菌数量,缩短发酵时间,提高生产效率,降低发酵成本,还可以提高酵母菌、芽孢杆菌和乳酸菌的保存时间,增强生物活性。专利CN201910605988.7则公开了植物乳酸菌在枯草芽孢杆菌固体菌剂保存中的应用及延长枯草芽孢杆菌的保存期的方法,能够有效抑制保存过程中枯草芽孢杆菌的活菌数下降,延长其保存周期。
鉴于现有技术中针对枯草芽孢杆菌和乳酸菌的微生物发酵饲料较多,因此,亟需提供一种乳酸菌和芽孢杆菌的发酵增效组合物,以进一步提高其发酵产物的微生物活菌数量,同时延长其保存时限。
发明内容
针对上述不足,本发明提供了一种乳酸菌和芽孢杆菌的发酵增效组合物。本发明所述的发酵增效组合物包括谷氨酸、苏氨酸、木犀草素、丹参素、硫酸锌、硫酸镁和D-甘露糖醇。将本发明所述的增效组合物添加到微生物发酵过程中能够提高发酵产品中的乳酸菌和芽孢杆菌的活菌数,同时提高发酵后产品中乳酸菌和芽孢杆菌的保存时限,具有良好的应用前景。
为了实现上述发明目的,本发明的技术方案如下:
一方面,本发明提供了一种乳酸菌和芽孢杆菌的发酵增效组合物,所述的发酵增效组合物包括谷氨酸、苏氨酸、木犀草素、丹参素、硫酸锌、硫酸镁和D-甘露糖醇。
具体地,所述的发酵增效组合物包括谷氨酸1-100mg/L、苏氨酸1-100mg/L、木犀草素1-100mg/L、丹参素1-100mg/L、硫酸锌1-50mg/L、硫酸镁1-50mg/L和D-甘露糖醇100-500g/L。
进一步具体地,所述的发酵增效组合物包括谷氨酸30-70mg/L、苏氨酸30-70mg/L、木犀草素30-70mg/L、丹参素30-70mg/L、硫酸锌20-40mg/L、硫酸镁20-40mg/L和D-甘露糖醇200-400g/L。
进一步具体地,所述的发酵增效组合物包括谷氨酸50mg/L、苏氨酸50mg/L、木犀草素50mg/L、丹参素50mg/L、硫酸锌30mg/L、硫酸镁30mg/L和D-甘露糖醇300g/L。
进一步具体地,所述的木犀草素与丹参素的比例为1:1-2,优选为1:1。
另一方面,本发明提供了上述发酵增效组合物在提高乳酸菌和芽孢杆菌发酵活菌数中的应用。
具体地,所述的乳酸菌为植物乳杆菌、粪肠球菌和/或动物双岐杆菌。
进一步具体地,所述的植物乳杆菌具有如下特性:厌氧或兼性厌氧,菌种为直或弯的杆状,单个、成对或成链状。表面菌落直径约3mm,凸起,呈圆形,表面光滑,细密,色白,偶尔呈浅黄或深黄色。
进一步具体地,所述的粪肠球菌具有如下特性:菌形圆或椭圆,菌落大而光滑,直径1-2mm,全缘,罕见色素。其营养要求低,在普通营养琼脂上也可生长。能在10℃或45℃pH9.6或含6.5%NaCl肉汤培养基中生长,并能耐65℃30min。
进一步具体地,所述的动物双岐杆菌具有如下特性:专性厌氧,菌落光滑,凸圆,边缘完整,乳脂呈白色,闪光并有柔软质地。最高生长温度为43-45℃。
具体地,所述的芽孢杆菌为枯草芽孢杆菌和/或地衣芽孢杆菌。
进一步具体地,所述的枯草芽孢杆菌具有如下特性:好氧菌,厌氧条件下生长不好,椭圆形芽孢,菌落表面粗糙不透明,菌落呈污白色或浅黄色。
进一步具体地,所述的地衣芽孢杆菌具有如下特性:好氧菌,芽孢呈椭圆形,菌落表面光滑,培养基含葡萄糖时,培养36h后菌落背面颜色呈红褐色,表面湿润,周边呈发散状,某些极少特异菌株(地衣亚种)代谢过程中会产生少量已酸和乳酸。
又一方面,本发明提供了上述发酵增效组合物在延长乳酸菌和芽孢杆菌发酵产品保存时限中的应用。
具体地,所述的乳酸菌为植物乳杆菌、粪肠球菌和/或动物双岐杆菌。
进一步具体地,所述的植物乳杆菌具有如下特性:厌氧或兼性厌氧,菌种为直或弯的杆状,单个、成对或成链状。表面菌落直径约3mm,凸起,呈圆形,表面光滑,细密,色白,偶尔呈浅黄或深黄色。
进一步具体地,所述的粪肠球菌具有如下特性:菌形圆或椭圆,菌落大而光滑,直径1-2mm,全缘,罕见色素。其营养要求低,在普通营养琼脂上也可生长。能在10℃或45℃pH9.6或含6.5%NaCl肉汤培养基中生长,并能耐65℃30min。
进一步具体地,所述的动物双岐杆菌具有如下特性:专性厌氧,菌落光滑,凸圆,边缘完整,乳脂呈白色,闪光并有柔软质地。最高生长温度为43-45℃。
具体地,所述的芽孢杆菌为枯草芽孢杆菌和/或地衣芽孢杆菌。
进一步具体地,所述的枯草芽孢杆菌具有如下特性:好氧菌,厌氧条件下生长不好,椭圆形芽孢,菌落表面粗糙不透明,菌落呈污白色或浅黄色。
进一步具体地,所述的地衣芽孢杆菌具有如下特性:好氧菌,芽孢呈椭圆形,菌落表面光滑,培养基含葡萄糖时,培养36h后菌落背面颜色呈红褐色,表面湿润,周边呈发散状,某些极少特异菌株(地衣亚种)代谢过程中会产生少量已酸和乳酸。
又一方面,本发明提供了上述发酵增效组合物在制备微生物发酵饲料中的应用。
具体地,所述的应用为在发酵前按照1-5%(v/w)的添加量,将上述发酵增效组合物添加至发酵固体培养基中。
进一步具体地,所述的发酵固体培养基包括:统糠或草粉50-60%、麦麸10-20%、玉米10-20%、饼粕5-15%、糖蜜或红糠1-5%,pH自然。
进一步具体地,所述的发酵固体培养基包括:统糠或草粉55%、麦麸16%、玉米15%、饼粕10%、糖蜜或红糠2%,pH自然。
又一方面,本发明提供了一种微生物发酵饲料的制备方法,所述的制备方法包括以下步骤:
(1)配制上述发酵增效组合物及固体培养基;
(2)将步骤(1)配制的发酵增效组合物加入到固体培养基中,混合均匀;
(3)取种子液接种于步骤(2)的固体培养基中发酵。
具体地,所述的种子液包括乳酸菌种子液和芽孢杆菌种子液。
进一步具体地,所述的乳酸菌种子液的培养方法为:从试管斜面挑取乳酸菌菌种,接种至MRS培养基,37℃静置培养22-26h,得到种子液。
进一步具体地,所述的芽孢杆菌种子液的培养方法为:从试管斜面上挑取芽孢杆菌菌种,接种至肉汤培养基,37℃,180rpm条件下振荡培养18-22h,得到种子液。
进一步具体地,所述的乳酸菌种子液和芽孢杆菌种子液的体积比为1:1。
具体地,所述的固体培养基包括:统糠或草粉50-60%、麦麸10-20%、玉米10-20%、饼粕5-15%、糖蜜或红糠1-5%,pH自然。
进一步具体地,所述的固体培养基包括:统糠或草粉55%、麦麸16%、玉米15%、饼粕10%、糖蜜或红糠2%,pH自然。
具体地,所述的步骤(2)中发酵增效组合物按照1-5%(v/w)的添加量加入到固体培养基中。
具体地,所述的步骤(3)中种子液按照1-10%的接种量接种于固体培养基中。
具体地,所述的步骤(3)中发酵温度为25-35℃,发酵时间为46-50h。
与现有技术相比,本发明的积极和有益效果在于:
本发明所述的发酵增效组合物包括谷氨酸、苏氨酸、木犀草素、丹参素、硫酸锌、硫酸镁和D-甘露糖醇。本发明首次发现将木犀草素和丹参素按照比例添加到增效组合物中,进一步应用于微生物发酵饲料制备中,可以提高微生物发酵饲料中乳酸菌和芽孢杆菌的活菌数同时提高发酵后产品中乳酸菌和芽孢杆菌的保存时限,具有良好的应用前景。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1一种发酵增效组合物
所述的发酵增效组合物包括谷氨酸50mg/L、苏氨酸50mg/L、木犀草素50mg/L、丹参素50mg/L、硫酸锌30mg/L、硫酸镁30mg/L和D-甘露糖醇300g/L。
实施例2一种发酵增效组合物
所述的发酵增效组合物包括谷氨酸50mg/L、苏氨酸50mg/L、木犀草素30mg/L、丹参素60mg/L、硫酸锌30mg/L、硫酸镁30mg/L和D-甘露糖醇300g/L。
实施例3一种发酵增效组合物
所述的发酵增效组合物包括谷氨酸30mg/L、苏氨酸30mg/L、木犀草素30mg/L、丹参素30mg/L、硫酸锌20mg/L、硫酸镁20mg/L和D-甘露糖醇400g/L。
实施例4一种发酵增效组合物
所述的发酵增效组合物包括谷氨酸70mg/L、苏氨酸70mg/L、木犀草素70mg/L、丹参素70mg/L、硫酸锌40mg/L、硫酸镁40mg/L和D-甘露糖醇200g/L。
实施例5一种发酵增效组合物
所述的发酵增效组合物包括谷氨酸50mg/L、苏氨酸50mg/L、木犀草素30mg/L、丹参素70mg/L、硫酸锌30mg/L、硫酸镁30mg/L和D-甘露糖醇300g/L。
实施例6一种发酵增效组合物
所述的发酵增效组合物包括谷氨酸100mg/L、苏氨酸100mg/L、木犀草素100mg/L、丹参素100mg/L、硫酸锌50mg/L、硫酸镁50mg/L和D-甘露糖醇500g/L。
实施例7一种发酵增效组合物
所述的发酵增效组合物包括谷氨酸50mg/L、苏氨酸50mg/L、硫酸锌30mg/L、硫酸镁30mg/L和D-甘露糖醇300g/L。
实验例1种子液制备
(1)枯草芽孢杆菌ATCC6633:从试管斜面上挑取枯草芽孢杆菌菌种,接种至肉汤培养基,37℃,180rpm条件下振荡培养20h,得到种子液。
(2)地衣芽孢杆菌ATCC11946:从试管斜面上挑取地衣芽孢杆菌菌种,接种至肉汤培养基,37℃,180rpm条件下振荡培养20h,得到种子液。
(3)植物乳杆菌ATCC8014:从试管斜面挑取植物乳酸菌菌种,接种至MRS培养基,37℃静置培养24h,得到种子液。
(4)粪肠球菌ATCC29212:从试管斜面挑取乳酸菌菌种,接种至MRS培养基,37℃静置培养22-26h,得到种子液。
(5)动物双岐杆菌ATCC25527:从试管斜面挑取乳酸菌菌种,接种至MRS培养基,37℃静置培养22-26h,得到种子液。
实验例2发酵固体培养基制备
所述的发酵固体培养基包括:统糠55%、麦麸16%、玉米15%、饼粕10%、红糠2%,pH自然。
实验例3一种微生物发酵饲料的制备方法及检测
(1)将任一实施例1-7配制的发酵增效组合物按照3%(v/w)的添加量加入到实验例2制备的固体培养基中,混合均匀;
(2)取实验例1制备的枯草芽孢杆菌种子液和植物乳杆菌种子液按照体积比1:1混合均匀,按照5%的接种量接种于步骤(1)的固体培养基中,发酵温度为30℃,发酵时间为50h,制备得到微生物发酵饲料。
(3)采用加速试验检测本申请所述的增效组合物的效果。将上述制备的微生物发酵饲料置于40±2℃、RH75±5%条件下进行6个月加速试验,在试验期间第0、1、3、6个月末取样检测其活菌数。
具体检测方法如下。
(1)供试液的制备
称取10g微生物发酵饲料样品,置于无菌均质袋内,加入pH7.2的磷酸盐缓冲液90g,用拍击式均质器拍打均匀60s,制成1:10的菌悬液。
(2)样品稀释
吸取1:10菌悬液1mL,沿管壁缓慢注入含有9mL磷酸盐缓冲液的无菌试管内(注意吸头尖端不要触及稀释液面),振摇试管使其混合均匀,制成1:100的菌悬液。这样依次稀释,分别得到10-3,10-4……10-9等浓度(每个稀释度必须更换无菌吸头)。
(3)接种混匀
乳酸菌接种:在无菌超净工作台中,采用无菌操作,吸取1mL不同稀释度菌悬液加入已灭菌的培养皿,将事先已灭菌并融化好的MRS培养基倒入上述培养皿中,每个约15-20mL。旋转混匀,静置凝固。每个样品取2-3个连续适宜稀释度,每一个稀释度重复2-3次,同时加磷酸盐缓冲液作对照空白。
芽孢杆菌接种:将稀释好的混合菌悬液放置在80℃的恒温水浴箱中30min,灭活去除乳酸菌类活菌,吸取1mL不同稀释度菌悬液加入已灭菌的培养皿,将事先已灭菌并融化好的NA培养基倒入上述培养皿中,每个约15-20mL。旋转混匀,静置凝固。每个样品取2-3个连续适宜稀释度,每一个稀释度重复2-3次,同时加磷酸盐缓冲液作对照空白。
(4)培养
将接种乳酸菌/芽孢杆菌的培养皿放置在恒温培养箱,盖好皿盖,平皿倒置,于37±1℃厌氧培养48±2h。
检测结果如下表1所示。
表1
由上述结果可知,在固体培养基中加入一定量的谷氨酸、苏氨酸、木犀草素、丹参素、硫酸锌、硫酸镁和D-甘露糖醇组成的增效组合物,能够明显增加微生物发酵饲料中的乳酸菌和芽孢杆菌的活菌数,同时提高发酵后饲料中乳酸菌和芽孢杆菌的保存时限。
实验例4一种微生物发酵饲料的制备方法及检测
(1)将实施例1配制的发酵增效组合物按照3%(v/w)的添加量加入到实验例2制备的固体培养基中,混合均匀;
(2)取实验例1制备的地衣芽孢杆菌种子液和粪肠球菌种子液、动物双岐杆菌种子液按照体积比1:1混合均匀,按照5%的接种量接种于步骤(1)的固体培养基中,发酵温度为30℃,发酵时间为50h,可制备得到微生物发酵饲料。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.一种发酵增效组合物在提高植物乳杆菌和枯草芽孢杆菌发酵活菌数,或延长植物乳杆菌和枯草芽孢杆菌发酵产品保存时限中的应用,其特征在于:所述的发酵增效组合物包括谷氨酸30-70mg/L、苏氨酸30-70mg/L、木犀草素30-70mg/L、丹参素30-70mg/L、硫酸锌20-40mg/L、硫酸镁20-40mg/L和D-甘露糖醇200-400g/L;所述的应用为在发酵前按照1-5%的添加量,将所述的发酵增效组合物添加至发酵固体培养基中。
2.根据权利要求1所述的应用,其特征在于:所述的发酵增效组合物包括谷氨酸50mg/L、苏氨酸50mg/L、木犀草素50mg/L、丹参素50mg/L、硫酸锌30mg/L、硫酸镁30mg/L和D-甘露糖醇300g/L。
3.根据权利要求1所述的应用,其特征在于:所述的木犀草素与丹参素的比例为1:1-2。
4.根据权利要求3所述的应用,其特征在于:所述的木犀草素与丹参素的比例为1:1。
5.一种微生物发酵饲料的制备方法,其特征在于:所述的制备方法包括以下步骤:
(1)配制权利要求1中所述的发酵增效组合物及固体培养基;
(2)将步骤(1)配制的发酵增效组合物加入到固体培养基中,混合均匀;
(3)取种子液接种于步骤(2)的固体培养基中发酵;所述的种子液包括植物乳杆菌种子液和枯草芽孢杆菌种子液。
6.根据权利要求5所述的制备方法,其特征在于:所述的植物乳杆菌种子液和枯草芽孢杆菌种子液的体积比为1:1。
7.根据权利要求6所述的制备方法,其特征在于:步骤(3)中所述的种子液的接种量为1-10%。
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