CN116948869B - 一株嗜酸乳杆菌fz1及其用于发酵五味子酒渣在替代抗生素中的应用 - Google Patents
一株嗜酸乳杆菌fz1及其用于发酵五味子酒渣在替代抗生素中的应用 Download PDFInfo
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- CN116948869B CN116948869B CN202310622204.8A CN202310622204A CN116948869B CN 116948869 B CN116948869 B CN 116948869B CN 202310622204 A CN202310622204 A CN 202310622204A CN 116948869 B CN116948869 B CN 116948869B
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- lactobacillus acidophilus
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- schisandra chinensis
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
- A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
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- C—CHEMISTRY; METALLURGY
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及生物技术领域,具体涉及一株嗜酸乳杆菌FZ1及其用于发酵五味子酒渣在替代抗生素中的应用,嗜酸乳杆菌FZ1于2023年4月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCCNo.27055,在模拟胃液3h后存活率可达到92.5±0.6%,45℃下存活率93.4±0.2%,明显高于其他突变菌株,且嗜酸乳杆菌FZ1耐胆盐能力及耐胃液酸能力显著高于原始菌株;且嗜酸乳杆菌FZ1具有抑制菌株的效果,嗜酸乳杆菌FZ1发酵得到的五味子酒渣饲料,纤维素、半纤维素含量显著减低,粗蛋白含量增高。木质素和多糖类无显著变化,抑制致病菌能力显著增强,是一种良好的抗生素替代品。
Description
技术领域
本发明涉及生物技术领域,具体涉及一株嗜酸乳杆菌FZ1及其用于发酵五味子酒渣在替代抗生素中的应用。
背景技术
五味子是木兰科五味子属植物的泛称,是一种药食同源的野生经济植物。果实不仅可入药,还可以加工成果酒、果汁饮料和保健品,是一种多功能、多用途的野生经济植物。五味子提取物中的多种活性成分均具有药理作用,如木脂素中的五味子素、五味子醇、五味子酚等活性成分具有神经保护、抗纤维化、抗癌、抗炎等特性。五味子酒渣是五味子酒及五味子饮料加工后后的副产物。酒渣营养丰富,含有粗蛋白质(CP)、粗纤维(CF)、粗脂肪(EE)等营养成分及钙、磷、铜、锌、铁、硒、镁等矿物元素,同时还富含维生素、黄酮、多糖、多酚等活性物质其作为饲料资源开发,不仅能缓解养殖业饲料资源不足的问题,还可降低养殖成本,提高经济效益,促进畜牧业及水果种植业、加工业等生态产业链的绿色可持续发展。但是果渣含水量大,不易贮存、运输,营养利用率低等问题多数被丢弃,造成资源浪费与环境污染。另外五味子酒渣还有部分木脂素类、多糖类、柠檬酸等生物活性成分,可以增强机体抗炎、抗氧化能力,调节肠道菌群平衡,改善机体代谢,且毒副作用较低,在动物养殖方面具有广阔的应用前景和研究价值。
在我国畜禽养殖业快速发展的大环境下,饲用抗生素的广泛使用在提高了生产力的同时,也造成了药物残留、耐药菌产生、动物肠道菌群紊乱、动物免疫功能下降等危害,甚至威胁人类的身体健康。微生物发酵五味子酒渣一方面可以减少果渣中纤维素、木质素、植酸、果胶、单宁等抗营养成分,同时提高其粗蛋白质、氨基酸等营养物质含量,改善果渣的营养结构和营养价值,提高其利用率;另一方面五味子酒渣中有效成分,可以提高微生物菌数,增加微生物抑制致病菌的能力。微生态制剂发酵五味子酒渣制备五味子渣发酵饲料可作为抗生素替代物运用于畜禽中。
嗜酸乳杆菌(Lactobacilus acidophilus)是一种重要的益生菌,其具有改善肠道健康、调节免疫、减轻肥胖、治疗肠道疾病等众多益生功能,并且由于益生菌的产酸功能,还可以替代饲料中的酸化剂,因此被认为是一种理想的微生态制剂。在医药、食品和饲料均有应用,市场需求巨大。
本发明在研究过程中意外的通过诱变选育得到了一株嗜酸乳杆菌FZ1,所述的嗜酸乳杆菌FZ1耐胆盐、耐胃酸的能力显著提高,且具有抑菌的效果,所述的嗜酸乳杆菌FZ1能够用于发酵饲料,具有广阔的应用前景。
发明内容
本发明的首要目的是提供一株嗜酸乳杆菌(Lactobacillus acidophilus)FZ1,所述的嗜酸乳杆菌FZ1于2023年4月10日,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.27055。
本发明的第二目的是提供一种菌剂,所述的菌剂包括所述的嗜酸乳杆菌FZ1。
本发明的第三目的是提供将所述的嗜酸乳杆菌FZ1或所述的菌剂通过冷冻干燥的方式制备成微胶囊菌剂。
本发明的第四目的是提供所述的嗜酸乳杆菌FZ1或所述的菌剂,所述的嗜酸乳杆菌FZ1具有下述性质:
(1)具有耐胆盐能力;
(2)具有耐酸能力;
(3)具有抑菌效果。
(4)耐高温特性。
本发明的第五目的是提供所述的嗜酸乳杆菌FZ1或所述的菌剂在制备发酵饲料中的应用。
本发明的第六目的是提供一种发酵饲料,所述的发酵饲料由所述的嗜酸乳杆菌FZ1对饲料原料发酵而得,所述的饲料原料为五味子酒渣。
本发明的第七目的是提供所述发酵饲料的制备方法,用所述的嗜酸乳杆菌FZ1对五味子酒渣发酵。
优选的,所述发酵的具体操作为:取五味子酒渣,豆粕粉,乳清粉混合均匀后加入权利要求1所述的嗜酸乳杆菌FZ1,压实覆盖或密封,室温下发酵后开封使用。
优选的,所述的五味子酒渣水分含量为30-40%。
优选的,所述的嗜酸乳杆菌FZ1加入的量为5g/kg。
本发明的有益效果是:本发明通过诱变选育得到了一株嗜酸乳杆菌FZ1,所述的嗜酸乳杆菌FZ1耐胆盐、耐胃酸的能力显著提高,且具有抑菌的效果,所述的嗜酸乳杆菌FZ1能够用于发酵饲料,具有广阔的应用前景。
附图说明
图1嗜酸乳杆菌FZ1菌落形态及革兰氏染色
A:菌落形态;B:革兰氏染色镜检
图2嗜酸乳杆菌FZ1抑制致病菌
A:大肠杆菌抑菌圈;B:金黄色葡萄球菌抑菌圈
图3五味子酒渣的制备工艺
具体实施方式
下面结合具体实施例对本发明的保护范围进行详细的阐述,应当说明的是,本发明所请求保护的保护范围并不受以下实施例的限制。
实施例一、嗜酸乳杆菌的诱变选育
1菌株来源及培养基
嗜酸乳杆菌(Lactobacillus acidophilus)La1分离于传统发酵泡菜中,保藏于甘肃省微生物菌种保藏中心(http://jzk.gsmsc.cn/),保藏号为CMCC 31201。菌株可抑制大肠杆菌和金黄色葡萄球菌。
模拟胃液:胃蛋白酶(1:10000)用灭菌后的PBS溶液配置为浓度为1mg/ml的胃蛋白酶溶液,用1mol/mL的HCl溶液调节pH 3.0后,经0.22μm微孔滤膜过滤除菌后备用。
2重离子束12C6+辐照诱变
将甘油法保藏的菌株嗜酸乳杆菌La1接种于MRS培养基,37℃恒温厌氧活化48h后,在无菌条件下将待辐照菌悬液1.5mL置于35mm无菌塑料平皿中,利用中国科学院近代物理所重离子加速器(Heavy ion research facility at Lanzhou,HIRFL)浅层辐照终端提供的80MeV/u,剂量率20Gy/min的12C6+重离子束进行辐照处理,辐照剂量分别为0、25、50、75、100、125、150、175Gy,每个剂量设置3个平行。按照不同辐照剂量依次菌株进行辐照,将辐照后菌液等量加入50%甘油,置于甘油管中-20℃冰箱保存。
3突变株的筛选
将不同辐照剂量的菌株涂布到含有0.3%胆盐的MRS平板中,37℃恒温静置培养48h,筛选可以明显生长的突变株。将突变活化后接入模拟胃液中3h后测定菌株的活菌数计算存活率。
4结果与分析
从表1中可以看出不同辐照剂量下的突变株相对于原始菌株都可以筛选得到在含0.3%胆盐MRS培养基中生长的菌株;筛选得到的菌株相对于原始菌株温度的耐受力明显提高。其中,突变株La-FZ1在模拟胃液3h后存活率可达到92.5±0.6%,明显高于其他突变菌株,且突变株La-FZ1耐胆盐能力及耐胃液酸能力显著高于原始菌株。其菌落形态如图1所示。
经过验证,所述的突变株La-FZ1为嗜酸乳杆菌,其拉丁名为Lactobacillusacidophilus,将其命名为嗜酸乳杆菌(Lactobacillus acidophilus)FZ1,保藏于保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.27055,保藏日期为2023年4月10日,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,联系方式为:010-64807355,传真:010-64807288,邮政编码:100101。
表1辐照诱变嗜酸乳杆菌突变株的筛选
应当说明的是,以下实施例中,所述的嗜酸乳杆菌(Lactobacillus acidophilus)FZ1简写为嗜酸乳杆菌FZ1。
实施例二、嗜酸乳杆菌FZ1抑菌特性
1.抑制致病菌的检测
采用琼脂牛津杯扩散法,筛选样品内具有抑制常见肠道致病菌的菌株。指示菌:大肠杆菌(Escherichia coli),金黄色葡萄球菌(Staphylococcus aureus),保藏于工业微生物菌种保藏中心甘肃分中心。
操作步骤:将MRS培养基冷却到70℃左右,向培养皿中加入20mL培养基,放凉15min后倒置;之后将倒置培养基平放,并在培养基表面准确滴加20μL新鲜培养的指示菌(108cfu/mL),用涂布棒涂布均匀。用火焰灼烧灭菌的镊子后,将经过高压蒸汽灭菌的牛津杯轻轻放置在培养基上,然后在牛津杯中加入200μL供试菌发酵液。加完样品后小心将平板放于培养箱,37℃培养24h。通过培养基中牛津杯周围透明抑菌圈的大小来判断抑菌性。
2.抑菌特性
嗜酸乳杆菌FZ1的抑菌效果图如图2所示,大肠杆菌抑菌圈直径3.5mm,金黄色葡萄球菌抑菌圈直径5.7mm,说明嗜酸乳杆菌FZ1具有抑制大肠杆菌和金黄色葡萄球菌的效果。实施例三、五味子酒渣饲料的制备
1.微生物菌剂的制备
微生物菌粉采用微胶囊及冷冻干燥连用的方式获得。海藻酸钠质量分数3.0%,聚丙烯酰胺质量分数为2.5%,固定化时间为2h,此条件下微胶囊包埋率达到85.7%。以可溶性淀粉、低聚木糖及谷氨酸钠复合配置而成的保护剂作为微胶囊的保护剂冷冻干燥效果最好。确定微胶囊冻干燥的最佳条件为:可溶性淀粉20%,低聚木糖9%,谷氨酸钠3%,脱脂乳粉10%,在真空度≦10Pa下,-50℃干燥48h,冻干微生物菌剂水分为0.18%,存活率达到87.3%。
2.五味子酒渣的制备工艺
五味子果酒、酒渣的制备工艺如图3所示。
3.五味子酒渣成分含量
五味子酒渣中纤维素、木质素含量采用纤维素测定仪测定。有机酸含量测定采用HPLC,KC-811离子色谱柱,柱温50℃,流动相为3mmol/LHCLO4溶液,进样量5μL。粗蛋白含量测定采用凯氏定氮仪测定。
木脂素的提取:五味子酒渣经60℃干燥2h后粉碎,采用超声波辅助乙醇提取的方法,在乙醇的体积分数为80%、温度85℃,超声时间30min,料液比1∶8的条件下回流提取2h,得五味子酒渣木脂素提取液,经旋转蒸发仪真空浓缩,冷冻干燥成粉,4℃冰箱密闭保存,备用。
多糖的提取:称取适量的五味子酒渣于100mL圆底烧瓶中,加入一定体积的去离子水,一定温度下加热处理一定的时间,提取液抽滤后浓缩至原体积的1/3,加入3倍量80%乙醇,4℃下醇沉过夜,离心后取适量沉淀物用蒸馏水定容于100mL容量瓶中,得样品溶液,除去蛋白质,透析48h,冷冻干燥备用。
4.五味子酒渣发酵饲料的制备
五味子酒渣为生产五味子酒副产物,水分含量为60-70%,晾晒至水分含量为30-40%待用。取水分含量为30-40%五味子酒渣100kg,100kg豆粕粉,乳清粉100kg混合均匀后按照5g/kg加入微生物菌粉,压实覆盖或密封即可,放置室温下(10-30℃)发酵2天后开封使用即可。
5.结果分析
五味子酒渣发酵前及加入豆粕粉、乳清粉及菌粉发酵后含量如表2所示。
表2五味子酒渣发酵前后成分含量及抑菌特性
五味子酒渣经发酵后纤维素、半纤维素含量显著减低,粗蛋白含量增高。木质素和多糖类无显著变化,抑制致病菌能力显著增强,是一种良好的抗生素替代品。
实施例四、五味子酒渣饲料在肉鸡养殖中替代抗生素的应用
1.试验设计
试验研究在饲料中添加五味子酒渣对肉鸡生长性能的影响,以及五味子酒渣饲料替代抗生素的效果。试验将180只1日龄健康爱拔益加(AA)肉仔鸡(1—42日龄)分为3个处理,每个处理6个重复,每个重复10只鸡。其中A组为饲喂基础饲粮组(按标准每千克饲料中添加有抗生素:维吉尼亚霉素20mg,硫酸抗敌素20mg),B组为在基础饲粮中添加五味子酒渣饲料20g/kg,C组为在基础饲粮中取出抗生素的同时添加五味子酒渣饲料20g/kg,试验周期共42d。
2生长指标的测定
试验期间记录各重复的耗料量,观察肉鸡的健康状况,记录全期死淘率,并于第42d以重复为单位空腹称重,计算平均日采食量、平均日增重和料重比,计算公式为:平均日采食量=耗料重/试验天数;平均日增重=(末重-初重)/试验天数;料重比=平均日采食量/平均日增重;死淘率=(死淘只数/原有总只数)×100%。
3结果分析
在肉鸡全期,在常规饲料基础上添加五味子酒渣饲料,料重比显著低于对照组,平均日增重显著高于对照组,平均日采食量无显著差异。无抗生素添加五味子酒渣饲料的肉鸡,各项指标与对照组无显著性差异。全期各实验组肉鸡死亡率未出现显著差异,说明在肉鸡养殖上五味子酒渣饲料在替代抗生素上起到一定效果。
表3不同配方下五味子酒渣饲料对肉鸡生长性能的影响
综上所述,本发明提供了一种嗜酸乳杆菌FZ1,所述的嗜酸乳杆菌FZ1保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.27055,保藏日期为2023年4月10日,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所;所述的嗜酸乳杆菌FZ1在模拟胃液3h后存活率可达到92.5±0.6%,明显高于其他突变菌株,且嗜酸乳杆菌FZ1耐胆盐能力及耐胃液酸能力显著高于原始菌株;且所述的嗜酸乳杆菌FZ1具有抑制菌株的效果,所述的嗜酸乳杆菌FZ1发酵得到的五味子酒渣饲料,纤维素、半纤维素含量显著减低,粗蛋白含量增高。木质素和多糖类无显著变化,抑制致病菌能力显著增强,是一种良好的抗生素替代品。
Claims (8)
1.一株嗜酸乳杆菌(Lactobacillus acidophilus)FZ1,其特征在于,所述的嗜酸乳杆菌FZ1于2023年4月10日,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.27055。
2.一种菌剂,其特征在于,所述的菌剂包括权利要求1所述的嗜酸乳杆菌FZ1。
3.如权利要求1所述的嗜酸乳杆菌FZ1或权利要求2所述的菌剂在制备发酵饲料中的应用。
4.一种发酵饲料,其特征在于,所述的发酵饲料由权利要求1所述的嗜酸乳杆菌FZ1对饲料原料发酵而得,所述的饲料原料为五味子酒渣。
5.权利要求4所述发酵饲料的制备方法,其特征在于,用所述的嗜酸乳杆菌FZ1对五味子酒渣发酵。
6.如权利要求5所述的发酵饲料的制备方法,其特征在于,所述发酵的具体操作为:取五味子酒渣,豆粕粉,乳清粉混合均匀后加入权利要求1所述的嗜酸乳杆菌FZ1,压实覆盖或密封,室温下发酵后开封使用。
7.如权利要求6所述的发酵饲料的制备方法,其特征在于,所述的五味子酒渣水分含量为30-40%。
8.如权利要求6所述的发酵饲料的制备方法,其特征在于,所述的嗜酸乳杆菌FZ1加入的量为5g/kg。
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