CN102851232B - 一株乳酸菌Lactobacillus parafarraginis ZH1及其应用 - Google Patents
一株乳酸菌Lactobacillus parafarraginis ZH1及其应用 Download PDFInfo
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- CN102851232B CN102851232B CN201210192264.2A CN201210192264A CN102851232B CN 102851232 B CN102851232 B CN 102851232B CN 201210192264 A CN201210192264 A CN 201210192264A CN 102851232 B CN102851232 B CN 102851232B
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- parafarraginis
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- Fodder In General (AREA)
Abstract
本发明公开了一株乳酸菌Lactobacillus parafarraginis ZH1及其应用。本发明所述乳酸菌保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地点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,保藏号为CGMCC NO.6079,保藏日期为2012年5月3日。本发明所述乳酸菌为格兰氏染色阳性杆菌,为葡萄糖异型发酵,耐酸,生长速度快,生育温域宽,高产苯甲酸和十六烷酸,可显著抑制青贮饲料的好氧变质。
Description
技术领域
本发明涉及微生物应用和饲料调制加工领域,具体涉及一株乳酸菌Lactobacillus parafarraginis ZH1及其在青贮饲料调制中的应用。
背景技术
青贮饲料颜色黄绿、气味酸香、柔软多汁、适口性好,在畜牧业生产中广泛应用,尤其在反刍动物饲养中,它已成为不可缺少的基础饲料。但青贮容器开封后,厌氧环境立即变成了有氧环境,好氧微生物开始增殖,从而使青贮饲料发生一系列的生物化学变化,常常导致青贮饲料发热、变质,即青贮饲料的好氧变质现象。通常情况下,好氧变质主要表现为温度上升和真菌菌斑的出现。对于不同的青贮饲料而言,好氧变质的速率不同。有的青贮饲料在开封后的数小时内则温度骤升,真菌菌斑产生;有的青贮饲料则能较长时间(1周或数周)保持不变。青贮饲料一旦发生好氧变质,饲料中的营养物质被氧化分解,干物质损失严重甚至全部不得不废弃;另外,一些有害微生物的活动会产生大量的有毒物质,轻则影响家畜健康、降低生产能力,重则造成中毒死亡,对畜牧业生产危害重大。
乳酸菌为革兰氏阳性菌,抗过氧化氢酶、厌氧、无孢子,无运动性,被认为是益生菌而广泛应用于青贮饲料生产当中。乳酸菌按发酵类型分为同型发酵乳酸菌和异型发酵乳酸菌。同型发酵乳酸菌利用水溶性碳水化合物(Water-soluble carbohydrates,WSC)产生较多的乳酸,改善青贮发酵品质,但抑制好氧变质的能力普遍较差。异型发酵乳酸菌利用WSC产生较多的乙酸,抑制了好氧性细菌、酵母和霉菌及青贮饲料好氧变质。目前,应用较广泛的商品菌剂是布氏乳杆菌(L. buchneri)。但其在某些条件下抑制青贮饲料的效果却不明显,如低温和短时间贮藏。
发明内容
本发明的目的在于根据现有技术中存在的上述不足,提供一株乳酸菌Lactobacillus parafarraginis ZH1。
本发明另一目的在于提供上述乳酸菌Lactobacillus parafarraginis ZH1的应用。
本发明上述目的通过以下技术方案予以实现:
一株类谷糠乳杆菌Lactobacillus parafarraginis (ZH1),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地点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,保藏号为CGMCC NO.6079,保藏日期为2012年5月3日。
本发明乳酸菌Lactobacillus parafarraginis ZH1的生物学特性为革兰氏染色阳性杆菌,葡萄糖异型发酵,耐酸性强(在pH4.0可以正常生长),生长速度快(MRS培养基培养24小时pH可降到4.5以下),生育温域宽(15-45℃可正常生长),在纯培养和青贮条件下高产苯甲酸和十六烷酸。
本发明乳酸菌Lactobacillus parafarraginis ZH1的获得方法是:富集培养该菌,稀释平板法筛选获得:将装有500 mL蒸馏水的锥形瓶、装有9 mL蒸馏水的试管、MRS固体培养基及平板灭菌,灭菌后再将MRS培养基倒入平板内。无菌条件下,取10 g青贮苏丹草样品于塑料袋内,再加入90 mL灭菌蒸馏水振荡均匀,使其终浓度为100 g·L-1,然后再取稀释后样品1 mL加入含有9 mL灭菌蒸馏水的试管振荡均匀,使其终浓度为10 g·L-1,此时取该液体0.03 mL反复进行平板划线分离,直至得到单菌落,将单菌落用接种针插入含有MRS固体培养基的试管中心,于4℃冰箱内保存。其中MRS培养基为:蛋白胨(Proteose peptone NO.3)10.0 g;牛肉膏(Beef extract)10.0 g;酵母提取物(Yeast extract)5.0 g;葡萄糖(Dextrose)20.0 g;吐温(Polysorbate 80)1 mL;柠檬酸铵(Ammonium citrate)2.0 g;醋酸钠(NaAc)5.0 g;硫酸镁(MgSO4·7H2O)0.1 g;硫酸锰(MnSO4·4H2O)0.05 g;磷酸氢二钾(K2HPO4)2.0 g;蒸馏水(H2O)1000 mL,固体培养基再加15 g/L琼脂(Agar),121℃,灭菌20 min。
本发明用生理生化方法筛选出高产苯甲酸和十六烷酸、耐酸及生长速度快的乳酸菌Lactobacillus parafarraginis ZH1。提取乳酸菌Lactobacillus parafarraginis全长基因,然后运用PCR 扩增引物 25f (SEQ ID NO:1) 和1492r (SEQ ID NO:2),扩增出16S rDNA基因,在NCBI上对比相关序列,确定其相似菌种。最后,将筛选出的菌株及对照菌(在国内使用最多的商品菌剂)分别添加到不同饲草中,开封后分析青贮产物和有氧稳定性并与对照菌进行对比,确认Lactobacillus parafarraginis ZH1可以显著提高青贮饲料的有氧稳定性。
本发明乳酸菌Lactobacillus parafarraginis ZH1可以用于调制青贮饲料。具体方法为:
(1)将待发酵饲料切断混合均匀;
(2)每千克待发酵饲料加入乳酸菌Lactobacillus parafarraginis ZH1 1.0×108 个以上;
(3)将饲料真空密封后贮藏。
作为一种优选方案,步骤(1)所述待发酵饲料切断至1~2cm。
作为一种优选方案,步骤(3)所述的贮藏温度为15~45℃,pH值为4.0~8.5。
与现有技术相比,本发明具有如下有益效果:
(1)本发明利用微生物乳酸菌Lactobacillus parafarraginis ZH1抑制青贮饲料好氧变质,成本低,安全,可靠,易于利用。
(2)本发明菌株可在我国各地使用,特别是低温贮藏条件下效果独特,克服了发酵品质好的青贮饲料易好氧变质的问题。
(3)本发明使用的乳酸菌Lactobacillus parafarraginis ZH1在低温环境下比市售乳酸菌添加剂效果更佳。
附图说明
图1为不同乳酸菌在MRS培养基中的十六烷酸产量示意图,其中柱间小写字母不同者为差异显著,P<0.05;ZH1,L. parafarraginis ZH1;ZH2,短乳杆菌(L. brevis);LP,植物乳杆菌(L. plantarum);LB,布氏乳杆菌(L. buchneri);
图2为不同乳酸菌在MRS培养基中的苯甲酸产量示意图,其中柱间小写字母不同者为差异显著,P<0.05;ZH1,L. parafarraginis ZH1;ZH2,短乳杆菌(L. brevis);LP,植物乳杆菌(L. plantarum);LB,布氏乳杆菌(L. buchneri);
图3为不同乳酸菌在晾晒黄燕麦青贮中苯甲酸和十六烷酸等的产量,其中柱间小写字母不同者为差异显著,P<0.05;CK,对照;ZH1,L. parafarraginis ZH1; LB,商品菌剂;BA,苯甲酸;TA,十四烷酸;PA,十六烷酸;OA,十八烷酸。
具体实施方式
以下结合实施例来进一步解释本发明,但实施例并不对本发明做任何形式的限定。
实施例1筛选高产苯甲酸和十六烷酸的优良乳酸菌
从苏丹草青贮饲料上分离的乳酸菌菌株ZH1,进行革兰氏染色和细胞形状观察,并根据生育温度(10、15、20、25、30、35、40、45、50℃)和生育pH(3.5、3.75、4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0)、MRS中产生苯甲酸和十六烷酸等生理生化实验。结果表明,ZH1菌株为革兰氏阳性,异型发酵的杆菌,在15-45℃和pH4.0-8.5条件下皆可生长,具有较强耐酸性(表1);可发酵大多数糖类(表2);在MRS中能够产生较多的苯甲酸和十六烷酸(图1和图2)。
表1 菌株ZH1的特性
项目 | 特性 |
革兰氏染色检测 | 阳性 |
过氧化氢酶活性 | 阴性 |
形状 | 杆状 |
发酵葡萄糖形式 | 异型 |
MRS中的生长pH | |
3.80 | - |
4.00 | + |
4.50 | + |
8.50 | + |
MRS中的生育温度 | |
15℃ | + |
40℃ | + |
45℃ | ± |
注:-,不生长;±,生长较弱;+,正常生长。
表2 菌株ZH1对不同糖的利用能力
糖类 | 利用性 | 糖类 | 利用性 |
对照 | - | 七叶灵 | + |
甘油 | - | 柳醇 | + |
赤癣醇 | - | 纤维二糖 | + |
D-阿拉伯糖 | - | 麦芽糖 | + |
L-阿拉伯糖 | + | 乳糖 | + |
核糖 | + | 蜜二糖 | + |
D-木糖 | - | 蔗糖 | + |
L-木糖 | - | 海藻糖 | + |
阿东醇 | - | 菊糖 | + |
β-甲基-D-木糖甙 | - | 松三糖 | + |
半乳糖 | + | 棉子糖 | + |
葡萄糖 | + | 淀粉 | - |
果糖 | + | 糖原 | - |
甘露糖 | + | 木糖醇 | - |
山梨糖 | - | 拢牛儿糖 | + |
鼠李糖 | - | D-松二糖 | + |
卫茅醇 | - | D-来苏糖 | - |
肌醇 | - | D-塔格糖 | - |
甘露醇 | + | D-岩糖 | - |
山梨醇 | + | L-岩糖 | - |
α-甲基-D-甘露糖甙 | + | D-阿拉伯糖醇 | + |
α-甲基-D-葡萄糖甙 | - | L-阿拉伯糖醇 | - |
N-乙酰-葡糖胺 | + | 葡萄糖酸盐 | + |
苦杏仁甙 | + | 2-酮基-葡萄糖酸盐 | - |
熊果甙 | + | 5-酮基-葡萄糖酸盐 | - |
注:-,不生长; +,正常生长。
乳酸菌ZH1的生理生化试验、培养基及其配制的方法如下:
1)革兰氏染色、发酵类型及形状观察参照东秀珠主编的《常见细菌系统鉴定手册》。
2)乳酸菌生长pH调节使用4 mol/L NaOH和4 mol/L HCl。
3)糖发酵采用API 50 CHL(bioMérieux, l’Etoile, France)分析。
4)培养基
MRS培养基(用于乳酸菌的培养):
蛋白胨(Proteose peptone NO.3)10.0 g;牛肉膏(Beef extract)10.0 g;酵母提取物(Yeast extract)5.0 g;葡萄糖(Dextrose)20.0 g;吐温(Polysorbate 80)1 mL;柠檬酸铵(Ammonium citrate)2.0 g;醋酸钠(NaAc)5.0 g;硫酸镁(MgSO4·7H2O)0.1 g;硫酸锰 (MnSO4·4H2O)0.05 g;磷酸氢二钾(K2HPO4)2.0 g。将上述试剂溶解,用蒸馏水定容至1000 mL,固体培养基再加15 g/L琼脂(Agar),121℃灭菌20 min。
API 50 CHL培养基:
蛋白胨(Proteose peptone NO.3)10.0 g;酵母提取物(Yeast extract)5.0 g;吐温(Polysorbate 80)1 mL;醋酸钠(NaAc)5.0 g;柠檬酸铵(Ammonium citrate)2.0 g;硫酸镁(MgSO4·7H2O)0.2 g;磷酸氢二钾(K2HPO4)2.0 g;硫酸锰(MnSO4·4H2O )0.05 g;溴甲酚紫(Bromcresol purple)0.17 g。将上述试剂溶解,用蒸馏水定容至1000 mL,分装在15 mL的试管中,每试管10 mL,121℃灭菌20 min。
实施例2 乳酸菌的鉴定
实施例1所得菌株在5 mL的MRS培养基中37℃培养过夜,菌液移入1.5 mL的离心管中,10000 rpm/min离心3 min~5 min收集菌,用TE0.1(10 mmol/L Tris–HCl, 0.1 mmol/L EDTA, pH 8.0)清洗两次,再用TIANamp Bacteria DNA Kit(TIANGEN BIOTECH CO., LTD, Beijing, China)试剂盒提取DNA,在OD600 nm处检测其吸光值。然后,进行PCR扩增,16S rDNA的扩增引物为25f (SEQ ID NO:1)和 1492r (SEQ ID NO:2),RCR反应为95℃(5 min)- 94℃(30 s)- 55℃(1 min)-72℃(1.5 min)-72℃(10 min),其中94℃(30 s)- 55℃(1 min)-72℃(1.5 min)反应循环30次。扩增产物送华大基因有限公司(中国)测序,结果在NCBI 的基因库上比对,找出与此菌亲缘相近的标准菌株玉米乳杆菌(Lactobacillus zeae RIA-482);干酪乳杆菌(Lactobacillus casei NM 108-1);副干酪乳杆菌(Lactobacillus paracasei subsp);鼠李糖乳杆菌(Lactobacillus rhamnosus NM 94-5);Lactobacillus parafarraginis NRIC 0680;并用DNAman软件分析,将筛选菌株的16S rDNA的部分序列(约1400 bp~1500 bp)与标准菌进行相似度分析,ZH1与Lactobacillus parafarraginis NRIC 0680相似度超过99%,应为同一种。
实施例3 添加到青贮饲料中的效果
以轻度晾晒和未晾晒的黄燕麦及玉米秸秆为材料进行了青贮添加试验。材料切断至1 cm~2 cm,混合均匀,每kg材料加入菌株ZH1约1.0×108 个。添加实施例2鉴定后的菌株ZH1后的材料装入30 cm×20 cm的聚乙烯青贮袋,每个处理装6袋,3袋45℃保存,3袋15℃保存,每袋约200 g,用真空密封机抽气、密封,黄燕麦青贮饲料置于15℃贮藏45 d,
低温15℃贮藏条件下,无添加的未晾晒黄燕麦和晾晒黄燕麦青贮饲料都产生了较多的乳酸和氨态氮,较少的乙酸,二者酵母较多,有氧稳定性低于40小时;添加布氏乳杆菌的黄燕麦青贮饲料也产生了较多的乳酸,较少的乙酸,其酵母也较多,有氧稳定性低于38小时,因此其对抑制黄燕麦青贮饲料好氧变质无效;添加ZH1的黄燕麦青贮饲料产生较少的乳酸,较多的乙酸,其酵母较少,有氧稳定性高于144小时,显著抑制了黄燕麦青贮饲料好氧变质(表3),说明ZH1在低温条件下抑制黄燕麦青贮饲料好氧变质的能力强于商品菌剂。另外,与对照和添加商品菌剂的晾晒黄燕麦青贮饲料相比,ZH1显著提高了该青贮饲料中的苯甲酸、十六烷酸、十四烷酸和十八烷酸的含量。苯甲酸和十六烷酸分别为1 g/kg DM和2 g/kg DM(图3),该含量的苯甲酸和十六烷酸对好氧细菌和酵母有较好的抑制效果。
在常温贮藏条件下,无添加的玉米秸秆青贮饲料产生了较多的乳酸和氨态氮,较少的乙酸,其好氧细菌较多,有氧稳定性为96小时;添加ZH1和商品菌剂减少了该青贮饲料乳酸和氨态氮及好氧细菌,增加了乙酸含量,有氧稳定性都超过了144小时,说明在常温贮藏条件下ZH1抑玉米秸秆青贮饲料的效果也较好,并与商品菌剂无差异。
营养成分及青贮发酵指标测定方法如下:
1)新鲜材料营养成分及微生物分析:干物质(DM)含量采用70℃干燥法测定,粗蛋白含量采用凯氏定氮法测定(定氮仪 KDN-103F,上海纤检仪器有限公司),WSC含量采用蒽酮-硫酸法测定,氨态氮含量用凯氏定氮仪直接蒸馏测定,缓冲能采用盐酸、氢氧化钠滴定法测定,乳酸菌、细菌、酵母菌和霉菌数量分别采用MRS(de-Man Rogosa Sharpe)琼脂培养基、营养琼脂培养基(Nutrient ager,广东环凯微生物有限公司)、马铃薯葡萄糖琼脂培养基(Potato-dextrose agar,广东环凯微生物有限公司)计数。乳酸菌用厌氧箱37℃培养2 d~3 d;细菌、酵母菌、霉菌用生化培养箱30℃培养2 d~4 d。
2)发酵品质分析:青贮袋开封后,取20g混匀的青贮料放入聚乙烯塑料封口袋中,加入80 mL 蒸馏水,在4℃下浸泡18 h后过滤,用pH计(PHS-3B,上海鹏顺科学仪器有限公司)测定浸提液pH值。乳酸菌、细菌、酵母菌和霉菌数量测定同鲜样相同,有机酸含量采用岛津LC-20AT型高效液相色谱仪测定:色谱条件:色谱柱(KC-811),流动相为3 mmol/L 的HClO4液,流速1 mL/min,柱温60℃,检测波长210 nm。
菌液和青贮饲料苯甲酸和十六烷酸检测方法如下:
苯甲酸和十六烷酸含量分析:取20 g接种ZH1的燕麦青贮饲料,加入80 mL蒸馏水,4℃下浸提18 h,然后用滤纸过滤得滤液。对于24小时培养的菌液,12000 rpm离心2分钟后,收集上清液。吸取6 mL滤液或上清液于C-18 SPE硅胶柱(迪马科技,上海)进行组分分离。分离方法为:
1)加入6 mL 95%乙腈水溶液对C-18 SPE柱进行活化。
2)活化后,加入6 mL 5%乙腈水溶液对C-18 SPE柱进行平衡。
3)加入6 mL 样品,然后用6 mL 5%乙腈水溶液对C-18 SPE柱进行洗脱,除去亲水性杂质。
4)加入6 mL 95%乙腈水溶液对C-18 SPE柱进行洗脱,并用1.5 mL离心管收集此洗脱液,每管收集1 mL。
在得到分离液后,采用岛津LC-20高效液相分析仪进行检测,得出色谱图。根据色谱图分析ZH1与对比菌间的差异,确定ZH1活性物质的色谱峰。条件为:色谱柱,Inertsil ODS-3 (GL Sciences, 日本);检测器,SPD-20A(岛津,日本),检测波长为210 nm;流动相,80% v/v乙腈水溶液;流速,1 mL/min;柱温,45℃;检测时间,6 min;进样量为20 μL。在确定ZH1活性物质色谱峰后,用岛津LC-20高效液相分析仪该色谱峰中的活性物质,进样量为100 μL,其它色谱条件同上。对收集的25 mL制备液,采用真空冷冻干燥仪(FD-1,北京博医康实验仪器有限公司)进行浓缩,得到约0.5 mL浓缩液。
用气质联用仪(GC-MS)(Agilent 5975C, 美国)对浓缩液进行检测并用苯甲酸和十六烷酸标准品作为内标进行定量分析。色谱条件为:色谱柱为Agilent DB-5ms,0.25 mm×30 m×0.25 um;载气He,流量10 mL/ min;进样口温度280℃;进样量1 μL;不分流;程序升温,100℃至270℃,升温20℃/min。质谱条件:电离源,电子电离(EI),电离电压70 eV;离子源温度230℃;扫描范围,10-700 amu(m/Z)。
名称:一株乳酸菌Lactobacillus parafarraginis ZH1及其应用
申请人:华南农业大学
SEQ ID NO:1 25f
AAC TGA AGA GTT TGA TCC TGG CTC
SEQ ID NO:2 1492r
TAC GGC TAC CTT GTT ACG ACT
Claims (1)
1. 乳酸菌Lactobacillus parafarraginis ZH1在提高青贮饲料中的苯甲酸、十六烷酸、十四烷酸和十八烷酸的含量中的应用;所述菌株ZH1于2012年5月3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,保藏号为CGMCC NO.6079。
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