CN112358996B - 一种用于提高构树青贮品质的干酪乳杆菌及其应用 - Google Patents
一种用于提高构树青贮品质的干酪乳杆菌及其应用 Download PDFInfo
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Abstract
本发明提供了一种用于提高构树青贮品质的干酪乳杆菌,该干酪乳杆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,保藏日期为2020年8月17日,保藏编号为CGMCC NO.20432。该干酪乳杆菌与菌株CGMCC NO.13132和CGMCC NO.16062的混合菌剂对构树进行青贮发酵制备青贮饲料。本发明提供的干酪乳杆菌具有高度协同性,抑制有害菌,产生大量乳酸,快速降低发酵料pH值。该菌株与CGMCC NO.13132、CGMCC NO.16062两株菌进行复配组合后的用于构树青贮饲料中,可快速产生乳酸,迅速降低构树青贮饲料pH值,抑制有害微生物生长繁殖,改善构树青贮品质,提高饲料的营养价值和适口性,且应用简易,成本低廉。
Description
技术领域
本发明属于微生物发酵饲料技术领域,具体涉及一种用于提高构树青贮品质的干酪乳杆菌及其应用。
背景技术
蛋白质饲料资源、优质粗饲料资源短缺是制约我国畜牧业发展的主要因素,开发新饲草资源是应对我国畜牧业发展的重要解决方案。构树是我国特有的桑科构树属落叶乔木,具有生长速度快、抗逆性极强,耐干旱、耐盐碱等特点,全株粗蛋白含量可达20%,树叶蛋白含量是苜蓿蛋白含量的1.4倍。然而,构树在雨季积累较多,且含水量高达70%,不便于长期存储,因此,采用青贮技术更适合构树长期保存利用。
青贮是通过密封压实创造封闭的厌氧环境,通过乳酸菌发酵产生乳酸等有机酸,使pH快速降低,抑制其他微生物的生长、代谢和繁殖,从而减少构树原料营养物质的损失,保护饲料免受不良微生物的侵害,改善饲料适口性,使其长期保存的方法。然而,构树叶两面均有绒毛分布,容易吸附空气中杂菌,原料上附着的乳酸菌数量少,缓冲能高等原因导致构树青贮效果不好。为了解决构树原料乳酸菌少、杂菌多等导致青贮失败的难题,需要额外添加乳酸菌剂,然而常规商品乳酸菌的添加不能改善构树青贮效果,且专用于构树青贮的干酪乳杆菌添加剂还未见报道。
发明内容
为了解决上述技术问题,本发明提供一种提高构树青贮品质的乳酸菌株,该株菌具有高度协同性,抑制有害菌,产生大量乳酸,快速降低发酵料pH值。
为实现上述目的,本发明采用以下的技术方案为:
一种用于提高构树青贮品质的干酪乳杆菌,该干酪乳杆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,保藏日期为2020年08月17日,保藏编号为CGMCCNO.20432。
用于提高构树青贮品质的复配青贮菌剂,该复配乳酸菌青贮菌剂包括编号为CGMCC NO.20432的干酪乳杆菌、和编号为CGMCC NO.13132的植物乳杆菌和CGMCC NO.16062的粪肠球菌。
如上所述的复配青贮菌剂,优选地,菌株CGMCC NO.13132:CGMCC NO.20432:CGMCCNO.16062的接种比例按体积比为0.5:2:0.5。
优选地,所述菌株CGMCC NO.13132和CGMCC NO.20432的菌浓度分别大于或等于108CFU/mL,所述CGMCC NO.16062的菌浓度大于或等于107CFU/mL。
用于提高构树青贮品质的干酪乳杆菌用于构树在制备青贮饲料中的应用,优选地,所述干酪乳杆菌与菌株CGMCC NO.13132和CGMCC NO.16062的混合菌剂对构树进行青贮发酵制备青贮饲料。
如上所述的应用,优选地,所述干酪乳杆菌与菌株CGMCC NO.13132和CGMCCNO.16062按体积比为2:0.5:0.5的进行混合。
如上所述的应用,优选地,所述干酪乳杆菌与菌株CGMCC NO.13132和CGMCCNO.20432的菌浓度分别大于或等于108CFU/mL,所述CGMCC NO.16062的菌浓度大于或等于107CFU/mL。
如上所述的应用,优选地,所述混合菌剂按新鲜构树枝叶总体积重量比的1%-2%添加到构树枝叶中,并添加构树枝叶质量的2%~5%的糖蜜,拌匀后置于呼吸袋中压实、密封,发酵温度为25℃~35℃青贮40~50天。
其中,体积重量比是指mL/g,1%~2%即100g中添加1~2mL菌液。
如上所述的应用,优选地,所述新鲜构树枝叶为1~2cm的长度。
本发明的有益效果在于:
本发明提供的一种用于提高构树青贮品质的干酪乳杆菌,具有高度协同性,抑制有害菌,产生大量乳酸,降低发酵料的pH值等优点的乳酸菌。将所筛菌种进行复配作为构树青贮乳酸菌接种剂,与中国微生物菌种保藏管理委员会普通微生物中心的CGMCCNO.13132、CGMCC NO.16062两株菌进行复配组合后的用于构树青贮饲料中,可快速产生乳酸,迅速降低构树青贮饲料pH值,抑制有害微生物生长繁殖,改善构树青贮品质,提高饲料的营养价值和适口性,且应用简易,成本低廉。
附图说明
图1为所筛菌株革兰氏染色的显微镜观察图;
图2为对筛选的三株菌株扩增16S rDNA的电泳图;
图3为三株乳酸菌生长曲线。
具体实施方式
以下实施例用于进一步说明本发明,但不应理解为对本发明的限制。在不背离本发明精神和实质的前提下,对本发明所作的修饰或者替换,均属于本发明的范畴。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1构树青贮专用菌种的筛选
1)液体富集:挑选pH 4.0-4.5的构树青贮样品,取3-5g样品放入灭菌后的100mLMRS液体培养基中,于37℃培养箱静置培养36h-48h。其中,MRS液体培养基由质量百分比为2%蔗糖、1%蛋白胨、1%牛肉膏、0.5%酵母浸粉、0.5%乙酸钠、0.2%柠檬酸铵、0.2%K2HPO4、0.05%MgSO4·7H2O、0.02%MnSO4·4H2O、0.1%吐温、余量为水的组分组成,pH调至6.2-6.4。
2)固体培养:吸取1mL上述液体富集的菌液用生理盐水进行10-4、10-5、10-6梯度稀释,吸取稀释液200μL均匀平板涂布于MRS固体培养基上,于37℃培养箱静置培养36h-48h,挑取菌落形态良好、长势较好的菌落于MRS固体培养基进一步划线纯化,同样培养条件,分别重复5-6次。其中,固体培养基成分与1步骤中的液体培养基成分相同,只是额外添加2%琼脂粉。
3)分离纯化:将经过固体培养的20株菌种分别进行10-4、10-5、10-6三个梯度稀释,分别取稀释后的菌液100μL涂布于碳酸钙选择性培养基中,于37℃培养箱静置培养36h-48h,比较菌落直径和水解圈直径,水解圈直径越大,则菌株的产酸能力越强,结果见表1。其中,固体培养基成分与步骤1中的液体培养基成分相同,额外添加2%碳酸钙。
4)革兰氏染色观察:用灭菌的接种环蘸取菌液置于干净的载玻片上,对菌种进行制片和革兰氏染色,对比所分离20株菌的形态变化。结果见表1,所挑选的20株菌种有17株菌涂片均呈现蓝紫色的革兰氏阳性菌,显微镜观察菌株为短棒状或链状,试验结果见图1。
5)过氧化氢酶实验:用灭菌的接种环蘸取菌液涂于干净的载玻片上,滴加30%过氧化氢,若有气泡产生则为过氧化氢实验阳性,否则则为阴性。实验结果表明,所选菌株的过氧化氢酶试验均为阳性,鉴定为乳杆菌属细菌。
表1乳酸菌鉴别生理生化实验
备注:+表示革兰氏染色呈蓝紫色,-表示过氧化氢试验结果阴性,/表示未检出。
6)16S rDNA菌种鉴定:通过革兰氏染色和过氧化氢酶实验,综合选育出三株菌落直径大、产酸能力强的乳酸菌BFEC-6、BFEC-7、BFEC-18进行琼脂凝胶电泳分析。采用细菌基因组DNA提取试剂盒提取基因DNA,通过PCR扩增细菌16S r DNA序列,其中菌株的PCR引物序列的上游引物P1和下游引物P2分别为:
P1(SEQ ID No.1):5’-AGAGTTTGATCCTGGCTCAG-3’;
P2(SEQ ID No.2):5’-GGTTACCTTGTTACGACTT-3’;
PCR循环结束后,PCR产物用0.1%的琼脂糖凝胶电泳分析,证明三株菌所提取的DNA基因组符合细菌属基因片段,电泳检测结果见图2。所提取的DNA基因组送北京诺赛基因组研究中心进行序列测定。
经16S r DNA引物进行扩增后,得到约1500bp左右的PCR扩增片段。将获得菌株的16S rDNA测序结果,序列具体见SEQ ID No.3,在NCBI数据库进行Blast序列比对,比对结果表明:菌株BFEC-7与干酪乳杆菌相似度≥97%,鉴定BFEC-7为干酪乳杆菌;菌株BFEC-6、BFEC-18与鼠李糖乳杆菌相似度≥97%,鉴定BFEC-6为鼠李糖乳杆菌。
通过革兰氏染色观察和过氧化氢酶试验共鉴定出17株外形为短棒状或链状的乳酸菌,结合菌落直径和水解圈直径挑选出BFEC-6、BFEC-7、BFEC-18三株菌进行16S rDNA菌种鉴定,鉴定菌株BFEC-7为菌落直径大、透明圈直径大、产酸能力强的干酪乳杆菌。将该菌株编号记为BFEC2002G3,并于2020年08月17日保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心,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,分类命名:干酪乳杆菌(Lactobacillus casei),保藏编号分别CGMCC NO.20432。
实施例2本发明的干酪乳杆菌的益生特性
将分离得到的干酪乳杆菌CGMCC NO.20432(BFEC-7)与CGMCC NO.13132的植物乳杆菌、CGMCC NO.16062的粪肠球菌分别接种于MRS液体培养基中,37℃培养箱静置培养,从第四小时起,每隔一小时采用酶标仪进行吸光度检测,记录吸光值评价菌株生长速度。实验数据见图3。其中,编号为CGMCC NO.13132的植物乳杆菌、CGMCC NO.16062的粪肠球菌在申请号为201911203375.7的“一种畜禽通用复合发酵菌剂及其制备方法和应用”中已有说明,在此不在赘述。本申请中所选用的菌株是经大量实验验证最后选择确定的三种菌株。
结论:CGMCC NO.20432和NO.13132菌株生长速度快,NO.16062菌株生长速度显著低于其它两株菌。
三株菌株(CGMCC NO.13132、NO.20432、NO.16062)按接种比例按体积比(其中CGMCC NO.13132和NO.20432菌液中的菌浓度分别为1×108CFU/mL,NO.16062菌液中的菌浓度1.1×107CFU/mL)为1:0:0;0:1:0;0:0:1;1:1:1;0.5:2:0.5;0.5:0.5:2;2:0.5:0.5进行菌悬液制备。菌悬液在37℃培养箱静置培养,同时检测pH、乳酸等指标,评价菌株产酸情况。实验数据见表2及表3。
表2菌悬液pH值
结论:三株菌复配培养后以NO.13132、NO.20432(BFEC-7即BFEC2002G3)、NO.16062的接种比例为0.5:2:0.5,其pH值低于其它各处理组,且显著低于单菌的菌悬液。
表3菌悬液产乳酸量(mmol/L)
结论:以NO.20432干酪乳杆菌发酵48h后乳酸产量最高;将三株菌进行复配以NO.13132、NO.20432、NO.16062的接种比例为0.5:2:0.5的复配混合菌悬液产乳酸量最高。
将斜面培养基上纯化后成熟的斜面菌苔用接种针转接于MRS液体培养基中,于37℃培养箱静置培养36h-48h,4℃条件下8000r/min离心10min,得乳酸菌上清液。三株菌(NO.13132、NO.20432、NO.16062)上清液的接种比例为按体积比1:0:0;0:1:0;0:0:1;1:1:1;0.5:2:0.5;0.5:0.5:2;2:0.5:0.5进行,其中NO.13132、NO.20432的菌浓度不低于108CFU/mL NO.16062菌浓度不低于107CFU/mL。取-80℃保存的大肠杆菌、金黄色葡萄球菌、沙门氏菌于37℃-38℃水浴快速化冻,按照2%-3%的接种量接种于LB液体培养基,37℃恒温培养18-24h,使液体培养基含菌量至108-1012CFU/mL。采用牛津杯法,将指示菌液100μL涂布于LB培养基表面,以无菌操作方法在平板表面放置牛津杯,再在杯中加入200μL不同接种比例的乳酸菌上清液,37℃静置培养8h,取出测量抑菌圈直径评价菌株抑菌效果。实验结果见表4。
表4菌体外抑菌结果(mm)
结论:以CGMCC NO.20432的干酪乳杆菌抑菌圈直径高于其它两株菌;将三株菌进行复配以0.5:2:0.5的比例抑菌圈直径最大,说明抑菌效果最佳。
实施例3构树青贮专用菌剂的青贮实验
将菌株CGMCC NO.13132、NO.20432、NO.16062的培养液(其中CGMCC NO.13132和NO.20432菌液中的菌浓度分别为108CFU/mL,NO.16062菌液中的菌浓度为107CFU/mL)的按体积比为0.5:2:0.5混合制成复配青贮菌剂,新鲜构树枝叶切割为1-2cm长度,在新鲜构树枝叶中按体积质量比加入(mL/g)为1%加入复配青贮菌剂,即100g鲜构树枝叶中加入复配青贮菌剂1.0mL,并添加构树枝叶质量的2%-5%的糖蜜。拌匀后置于呼吸袋中压实、密封,25℃-35℃青贮40-50天,并于青贮实验的第0、5、10、20、30、40天取青贮饲料,使用pH计检测pH值,利用高效液相色谱测定培养液中乳酸含量。
对比例1
对比例1与实施例3的区别在于将“复配青贮菌剂+糖蜜”替换为“等体积蒸馏水”。
对比例2
对比例2与实施例3区别仅在于,将“复配青贮菌剂+糖蜜”替换为“青贮传奇+糖蜜”。(其中,青贮传奇来自于内蒙古青贮传奇科技有限公司)使用pH计检测pH值,利用高效液相色谱测定培养液中乳酸含量。实验数据见表5和表6。
表5构树青贮饲料pH变化
表6构树青贮饲料乳酸含量变化(mmol/L-1)
结论:青贮结束后,观察对比例1及对比例2和实施例3处理后的构树饲料,实施例3处理组的构树青贮饲料pH值下降最快,青贮40d后乳酸产量最高。说明本发明筛选出的复合青贮菌剂对构树青贮效果优于现有复合菌剂。
序列表
<110> 博益德(北京)生物科技有限公司
<120> 一种用于提高构树青贮品质的干酪乳杆菌及其应用
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<170> SIPOSequenceListing 1.0
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tacggctccg gcttcgggtg 20
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<213> 人工序列(Artificial Sequence)
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tgtcacgttc gtggcaagta c 21
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<211> 1393
<212> DNA
<213> 人工序列(Artificial Sequence)
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tacggctccg gcttcgggtg ttacaaactc tcatcgtgtg acgggcggtg tgtacatggc 60
ccgggaacgt attcaccgcg gcgtgctgat ccgcgttaac tagcgattcc gacttcgtgt 120
aggcgagttg cagccatcag tccgaactga gaatggcttt aagagattag cttgacctcg 180
cggtctcgca actcgttgta cctaccattg tagcacgtgt gtagcccagg tcatatgggg 240
catgatgatt tgacgtcatc ccgaccttcc tccggtttgt caccggcagt cttactagag 300
tgccctactg aatgctggca actagtctaa aggcttgcgc tcgttgcggg acttaaccca 360
acatctcacg acacgagctg acgacaacca tgcaccacct gtcattttgc ccccgaaggg 420
gaaacctgat ctctcaggtg atcaaaagat gtcaagacct ggtaaggttc ttcgcgttgc 480
ttcgaattaa accacatgct ccaccgcttg tgccggcgcc cgtcaaatcc tttgagtttc 540
aaccttgcgg tcgaactccc caggcggaat gcttaatgcg ttagctgcgg cactgaaggg 600
cggaaaccct ccaacaccta gcattcatcg ttaacggcat ggactaccag ggtatctaat 660
cctgttcgct acccatgctt tcgagcctca gcgtcagtta cagaccagac agccgccttc 720
gccactggtg ttcttccata tatctacgca tttcaccgct acacatggag ttccactgtc 780
ctcttctgca ctcaagtttc ccagtttccg atgcgcttcc tcggttaagc cgagggcttt 840
cacatcagac ttaaaaaacc gcctgcgctc gctttacgcc caataaatcc ggataacgct 900
tgccacctac gtattaccgc ggctgctggc acgtagttag ccgtggcttt ctggttggat 960
accgtcacgc cgacaacagt tactctgccg accattcttc tccaacaaca gagttttacg 1020
acccgaaagc cttcttcact cacgcggcgt tgctccatca gacttgcgtc cattgtggaa 1080
gattccctac tgctgcctcc cgtaggagtt tgggccgtgt ctcagtccca atgtggccga 1140
tcaacctctc agttcggcta cgtaacatcg ccttggtgag ccgttacctc accaactagc 1200
taatacgccg cgggtccatc caaaagcgat agcttgcgcc atctttcagc caagaaccat 1260
gcggttcttg gatttatgcg gtattagcat ctgtttccaa atgttatccc ccacttaagg 1320
gctggttacc cacgtcttac tcacccgtcc gccactcgtt ttaagttgaa tcacagtgca 1380
agcaccgttc atg 1393
Claims (10)
1.一种用于提高构树青贮品质的干酪乳杆菌,该干酪乳杆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,保藏日期为2020年08月17日,保藏编号为CGMCC NO.20432。
2.用于提高构树青贮品质的复配青贮菌剂,其特征在于,该复配青贮菌剂包括编号为CGMCC NO.20432的干酪乳杆菌、和编号为CGMCC NO.13132的植物乳杆菌和CGMCC NO.16062的粪肠球菌。
3.根据权利要求2所述的复配青贮菌剂,其特征在于,菌株CGMCC NO.13132:CGMCCNO.20432:CGMCC NO.16062的接种比例按体积比为0.5:2:0.5。
4.根据权利要求3所述的复配青贮菌剂,其特征在于,所述菌株CGMCC NO.13132和CGMCC NO.20432的菌浓度分别大于或等于10 8CFU/mL,所述CGMCC NO.16062的菌浓度大于或等于10 7CFU/mL。
5.权利要求1所述的用于提高构树青贮品质的干酪乳杆菌用于构树在制备青贮饲料中的应用。
6.根据权利要求5所述的应用,其特征在于,所述干酪乳杆菌与菌株CGMCC NO.13132和CGMCC NO.16062的混合菌剂对构树进行青贮发酵制备青贮饲料。
7.根据权利要求6所述的应用,其特征在于,所述干酪乳杆菌与菌株CGMCC NO.13132和CGMCC NO.16062按体积比为2:0.5:0.5的进行混合。
8.根据权利要求7所述的应用,其特征在于,所述干酪乳杆菌与菌株CGMCC NO.13132和CGMCC NO.20432分别大于或等于10 8CFU/mL,所述CGMCC NO.16062的菌浓度大于或等于107CFU/mL。
9.根据权利要求6所述的应用,其特征在于,所述混合菌剂按新鲜构树枝叶总体积重量的1%-2%添加到构树枝叶中,并添加构树枝叶质量的2%~5%的糖蜜,拌匀后置于呼吸袋中压实、密封,发酵温度为25℃~35℃青贮40~50天。
10.根据权利要求9所述的应用,其特征在于,所述新鲜构树枝叶为1~2cm的长度。
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