CN111534459A - 一株高产淀粉酶的发酵乳杆菌及其在制备发酵饲料中的应用 - Google Patents
一株高产淀粉酶的发酵乳杆菌及其在制备发酵饲料中的应用 Download PDFInfo
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- CN111534459A CN111534459A CN202010287795.4A CN202010287795A CN111534459A CN 111534459 A CN111534459 A CN 111534459A CN 202010287795 A CN202010287795 A CN 202010287795A CN 111534459 A CN111534459 A CN 111534459A
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- lactobacillus fermentum
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- fermented
- fermented feed
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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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Abstract
本发明属于微生物技术领域,具体公开了一株高产淀粉酶的发酵乳杆菌及其在制备发酵饲料中的应用。该菌株对模拟胃液、胆盐以及人工肠液都有较强的耐受,且该菌株产酸迅速,对大肠杆菌和金黄色葡萄球菌有抑制作用,显示出较强的益生特性。此外该菌株还能产淀粉酶,水解利用淀粉产乳酸,表现较好的谷物发酵潜力。该菌株已保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M2020067。利用该菌株可直接以麸皮、玉米和豆粕为主要原料的底物进行发酵,制备的发酵饲料应用在小龙虾和生猪养殖上能显著提高生产性能,具有较好的应用前景。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一株高产淀粉酶的发酵乳杆菌及其在制备发酵饲料中的应用。
背景
畜牧业中抗生素的广泛使用甚至滥用导致微生物产生耐药性、宿主产生过敏反应、肉制品中抗生素残留、环境污染等一系列问题,严重威胁畜牧业的长远发展和人类健康,所以寻找无毒、无残留、应用效果好的饲料添加剂来替代抗生素意义重大。益生菌通过调节宿主体内的微生物生态平衡,可抑制病原菌的生长,保护宿主免受病原菌的感染,是抗生素的理想替代品。发酵饲料是指利用一些特种功能性微生物与常规的饲料原料及辅料混合后发酵,最终制成的含有一定量活性益生菌、安全无污染的饲料。饲料经发酵后具有适口性增强、抗营养因子降低、消化吸收率提高、增强动物免疫力、促进动物生长等优点,是无抗养殖重要的技术手段之一。
乳酸菌是一类能够快速发酵碳水化合物,并以乳酸为主要代谢产物的革兰氏阳性细菌,是使用最广泛的益生菌,自然定植于动物体的消化道中,是动物肠道中的正常菌群。乳酸菌可以通过粘附素与肠粘膜细胞紧密结合,在肠粘膜表面定植占位;还能产生多种抑菌因子,抑制有害菌繁殖;进而改善肠道内菌群结构,提高动物机体免疫力;保持动物健康,提高生产能力。
发酵乳杆菌在我国已被批准作为饲料添加剂,但是其在养殖端尚未被广泛使用。据报道,发酵乳杆菌通过调控肠道屏障和免疫相关基因的表达,可以增强感染产气荚膜梭菌肉鸡肠道屏障和免疫功能,促进后肠乳酸菌属的增殖,减轻肠道损伤(夏亿;2019);发酵乳杆菌I5007有促进新生仔猪肠道发育,增强肠道功能,降低新生仔猪肠道梭菌数量作用(Hong;2014);饮水饲喂发酵乳杆菌-F6可促进肉鸡小肠绒毛发育,增强肉鸡肠道功能,提高采食量,提高日增重和饲料粗蛋白消化率,促进肉鸡生长(付果花;2010)。可见在饲料中添加发酵乳杆菌可以改善动物生长性能、维持宿主肠道微生态平衡、增强机体免疫力的作用。利用发酵乳杆菌制备发酵饲料不仅实现了饲料预消化,提升了饲料的营养价值,还保留有大量乳酸菌生长代谢产物以及部分活菌,可以进一步增强动物健康,提高生产水平,具有很好的市场应用前景。
目前应用的乳酸菌多是以前科研工作者所筛选用于乳制品的发酵菌株,很少有适合于以谷物为主要原料进行发酵的乳酸菌菌株,而发酵饲料的主要原料是玉米麸皮等谷物,因此筛选具有水解淀粉能力的乳酸菌,对实现谷物的直接发酵,简化生产工艺,降低发酵饲料生产成本,提升发酵饲料品质具有重要意义。此外,乳酸菌后发酵阶段需要有还原糖以维持其活性,能利用淀粉发酵的乳酸菌在发酵饲料保存和后发酵阶段更能维持活性。另外发酵饲料一般为生料发酵,需要乳酸菌快速产酸并增殖以抑制原料中较多的杂菌生长,保证发酵饲料的品质。因此,在发酵饲料中选用能直接利用谷物淀粉快速发酵产酸的乳酸菌有助于降低生产成本,维持乳酸菌活性并稳定发酵饲料品质。
发明内容
本发明的目的在于提供一株高产淀粉酶的乳酸菌,该菌株为发酵乳杆菌(Lactobacillus fermentum)F36,保藏编号为CCTCC NO:M2020067。
本发明的另一个目的在于提供了一株高产淀粉酶的乳酸菌的应用,所述的乳酸菌可用于制备L-乳酸,生产淀粉酶,以及制备发酵饲料。
为了达到上述目的,本发明采取以下技术措施:
申请人自黄酒糟中分离得到一株乳酸菌,经16S rDNA基因序列鉴定为发酵乳杆菌,该菌株已于2020年4月9日送至中国典型培养物保藏中心保藏,分类命名:发酵乳杆菌(Lactobacillus fermentum)F36,保藏编号为:CCTCC NO:M2020067,地址:中国武汉武汉大学。
所述发酵乳杆菌(Lactobacillus fermentum)F36为革兰氏阳性杆菌,无芽孢。在MRS固体培养基上菌落为乳白色、表面光滑湿润、圆形、隆起、边缘整齐,直径大小一般为1.0-2.5mm。菌株经简单染色后在显微镜下呈杆状。利用葡萄糖异型乳酸发酵产L-乳酸;最适生长pH为6.0-6.5;最适生长温度为37℃。
发酵乳杆菌(Lactobacillus fermentum)F36在制备水产动物饲料添加剂中的应用;包括将该菌株作为饲料添加剂直接与水产动物饲料混合;或者将该菌株固态发酵后,将固态发酵产物一起与水产动物饲料混合。
发酵乳杆菌(Lactobacillus fermentum)F36在制备畜禽饲料添加剂中的应用;包括将该菌株作为饲料添加剂直接与畜禽饲料混合;或者将该菌株固态发酵后,将固态发酵产物一起与畜禽饲料混合。
以上所述的应用中,优选的,固态发酵的步骤包括:
40-60份麸皮、10-30份玉米、15-30份豆粕、3-8份石粉、0.05-1份酸性蛋白酶、0.05-1份中性蛋白酶、0.05-0.2份枯草芽孢杆菌、60份水以及10-20份发酵种子液混合均匀后30-37℃条件下厌氧发酵;以上所述的份数均为重量份;
所述的酸性蛋白酶和中性蛋白酶的酶浓度均为5万U/g;
所述的枯草芽孢杆菌的有效活菌数为2×1010CFU/g;
所述的发酵饲料种子液的制备:5-10份麸皮、1-2份豆粕加90份水灭菌降温后接种发酵乳杆菌F36种子液(接种量106-107CFU/ml)和耐高温酿酒酵母(接种量104-105CFU/ml),30-37℃条件下静置发酵即得。
以上所述的麸皮,玉米,豆粕的含水量为10-14%。
所述的耐高温酿酒酵母要求酵母37℃条件下存活率100%且能增殖,40℃条件下存活率在80%以上。
发酵乳杆菌(Lactobacillus fermentum)F36在制备L-乳酸中的应用;或者用于制备淀粉酶,也属于本发明的保护范围。
与现有技术相比,本发明具有以下优点:
本发明提供的发酵乳杆菌(Lactobacillus fermentum)F36具有耐酸、耐胆盐,产酸速率快,抑制大肠杆菌和金黄色葡萄球菌等生物学特性,尤其与众不同的是该发酵乳杆菌F36可表达淀粉酶,具有水解利用淀粉的能力,在含淀粉培养基中表达的淀粉酶酶活可达238U/ml,利用葡萄糖产酸速率可达2.53g/L/h,L-乳酸纯度98%。
本发明提供的发酵乳杆菌(Lactobacillus fermentum)F36可直接发酵谷物饲料,有很好的发酵饲料制备优势。原料无需蒸煮熟化,亦无需添加糖化酶和葡萄糖或者蔗糖等小分子糖类物质。产品口感酸香,风味好,酸度高,发酵乳杆菌活菌数高,发酵结束后活菌数可达1×1010CFU/g,两个月储存期后乳酸菌活菌数仍有2×109CFU/g以上。
附图说明
图1为发酵乳杆菌F36平板菌落形态图。
图2为发酵乳杆菌F36简单染色镜检形态图。
图3为发酵乳杆菌F36发酵饲料制备时物质变化图。
图4为发酵乳杆菌F36发酵饲料储存周期内物质变化图。
具体实施方式
本发明所述技术方案,如未特别说明,均为本领域的常规方案;所述试剂或材料,如未特别说明,均来源于商业渠道。
实施例1:
发酵乳杆菌F36菌株的筛选:
1.培养基的配制:
筛选培养基:蛋白胨5g/L;酵母膏3g/L;葡萄糖1g/L;淀粉5g/L;琼脂20g/L;0.5%溴甲酚紫10ml/L;pH值6.8-7.0。
MRS液体培养基:葡萄糖20g/L;蛋白胨10g/L;酵母提取物5g/L;牛肉浸粉10g/L;柠檬酸二铵2g/L;醋酸钠5g/L;吐温80 1g/L;磷酸氢二钾2g/L。
YG液体培养基:葡萄糖20g/L;酵母浸粉5g/L;碳酸钙10g/L。
固体培养基在液体培养基的基础上添加1.5%的琼脂,所有培养基115℃灭菌20min。
2.乳酸菌初筛:
取黄酒糟、黄酒发酵液、发酵玉米面等样品各5g置于100ml生理盐水中振荡30min,取1ml进行梯度稀释,涂布筛选培养基平板,37℃培养72h后挑选典型乳酸菌菌落(菌落周边变黄且有分解淀粉的透明圈)进行划线分离培养,共挑选30个,MRS斜面保藏。
3.分解淀粉乳酸菌复筛:
将初筛得到的菌株转接到100ml MRS液体培养基37℃培养18h,再各转接15ml到300ml YG液体培养基中,37℃培养6h后取样检测葡萄糖和乳酸含量,并计算产酸速率。另外配置含淀粉的MRS培养基(将葡萄糖的80%用淀粉替代),接种上述10%的乳酸菌液后37℃培养72h,取样检测乳酸菌活菌数以及淀粉酶酶活。
结果显示:8株菌株产乳酸速率在2.0g/L/h以上。3株菌株淀粉酶酶活在200U/ml以上,且发酵液中乳酸菌活菌数都超过109CFU/ml,酶活和菌数都高于其它菌株。其中只有菌株F36符合两组条件,其产乳酸速率达到2.53g/L/h,乳酸产率0.65g/g,且L-乳酸纯度98%以上,表明该菌株异型发酵产L-乳酸;其乳酸活菌数为2.34×109CFU/m,淀粉酶酶活为238U/ml。
4.菌株F36鉴定:
用结晶紫对筛选出来的目的菌株F36进行简单染色,并通过光学显微镜进行观察。发现该菌株为革兰氏阳性杆菌,无芽孢。在MRS固体培养基上菌落为乳白色、表面光滑湿润、圆形、隆起、边缘整齐,直径大小一般为1.0-2.5mm。菌株经简单染色后在显微镜下呈杆状。利用葡萄糖异型乳酸发酵产L-乳酸;最适生长pH为6.0-6.5;最适生长温度为37℃。
挑取F36单菌落用通用引物进行扩增,对扩增产物进行16S rDNA测序分析。结果表明该序列与Lactobacillus fermentum 16S rDNA序列的相似性最高,达99%,可确定该菌株为发酵乳杆菌,命名为发酵乳杆菌F36。该菌株已于2020年4月9日送至中国典型培养物保藏中心保藏,分类命名:发酵乳杆菌(Lactobacillus fermentum)F36,保藏编号为:CCTCCNO:M2020067,地址:中国武汉武汉大学。
实施例2:
发酵乳杆菌F36益生性能试验:
取MRS培养基中生长至对数后期的发酵乳杆菌F36种子液离心,用0.9%的生理盐水洗涤两次后重新悬浮,以108CFU/ml的量接种到pH2.5的人工胃液中,于37℃水浴锅中孵育培养,每隔1h取样稀释涂YPD平板测定活菌数。
结果显示该发酵乳杆菌F36耐受pH 2.5的模拟胃液1h时,存活率97.20%;2h时,存活率87.35%。
取生长至对数后期的发酵乳杆菌F36种子液离心,用0.9%的生理盐水洗涤两次后重新悬浮,以108CFU/ml的量接种到人工肠液中,于37℃水浴锅中孵育培养8h取样稀释涂YPD平板测定活菌数。
结果显示该发酵乳杆菌F36在人工肠液8h处理存活率为66.32%。
取生长至对数后期的发酵乳杆菌F36种子液离心,用0.9%的生理盐水洗涤两次后重新悬浮,以108CFU/ml的量接种到0.1%-0.5%的猪胆盐环境中,于37℃水浴锅中孵育培养6h取样稀释涂YPD平板测定活菌数。
结果显示该发酵乳杆菌F36在0.1%-0.5%的猪胆盐环境中,该发酵乳杆菌的存活率随着猪胆盐浓度升高而逐渐降低。在猪胆盐浓度0.1%时37℃孵育6h,存活率为83.67%;在猪胆盐浓度0.2%时37℃孵育6h,存活率为78.58%;在猪胆盐浓度0.3%时37℃孵育6h,存活率为47.50%;在猪胆盐浓度0.4%时37℃孵育6h,存活率为38.53%;在猪胆盐浓度0.5%时37℃孵育6h,存活率为35.56%。
发酵乳杆菌F36于MRS液体培养基中静置培养12h备用。用乳酸调MRS培养基的pH值使其与乳酸菌发酵液pH值相同,将其作为对照(CK)。将0.1mL大肠杆菌K88、金黄色葡萄球菌ATCC 27217培养液分别均匀涂布在固体LB培养基上,待平板表面干后用无菌镊子取牛津杯(内径6mm,外径8mm)紧贴于培养基表面,在各牛津杯中分别加入200uL乳酸菌发酵液和对照液,37℃培养24h,观察有无抑菌圈并测定对照与乳酸菌发酵液抑菌圈的直径。
结果显示发酵乳杆菌F36发酵液的抑菌圈直径明显大于对照组,说明该菌株对大肠杆菌K88和金黄色葡萄球菌ATCC 27217均有明显抑制作用。
表1分离菌株的抑菌试验结果
注:各组与对照组比较,**表示差异极显著(P<0.01)
实施例3:
发酵乳杆菌F36发酵饲料的制备:
发酵乳杆菌F36种子液制备:在YPD液体培养基中接种5%(体积比)乳酸菌后于37℃静置培养24h得到乳酸菌种子液。YPD培养基:1%酵母浸粉,2%蛋白胨,2%葡萄糖。
发酵饲料种子液的制备:10份麸皮、2份豆粕加90份水(以上份数均为重量份)后升温至121℃灭菌20min,降温至37℃后接种发酵乳杆菌F36种子液(接种量2×106CFU/ml)和耐高温酿酒酵母(接种量2×104CFU/ml),37℃条件下静置发酵24h得到发酵饲料种子液。测得此种子液中乳酸菌活菌数7.8×108CFU/ml,酵母活菌数3.2×107CFU/ml。
发酵饲料生产方法:50份麸皮、20份玉米、25份豆粕、5份石粉、0.05份酸性蛋白酶(酶活5万U/g)、0.05份中性蛋白酶(酶活5万U/g)、0.05份枯草芽孢杆菌(菌数2×1010CFU/g)、60份水以及10份上述发酵饲料种子液(以上所述的份数均为重量份)混合均匀后装入呼吸袋或者呼吸桶中,30℃条件下厌氧发酵7d,既得发酵乳杆菌F36发酵饲料。
7d后检测该发酵饲料乳酸菌活菌数为:乳酸菌1.05×1010CFU/g,酵母5.56×107CFU/g;酸度10.45%,乳酸含量6.62%,乙酸含量3.31%,pH 4.75。
以上所述的麸皮、豆粕、玉米的含水量分别是12.5%、11%和10%。
实施例4:
发酵乳杆菌F36大型发酵生产及储存过程中物质变化:
按实施例3所述方法放大10倍制备发酵饲料,在发酵7d内每天取样,7d发酵结束后置于20℃条件下储存9周,每周取样,检测pH、酸度、活菌数、乳酸和乙酸含量。
7d发酵周期内物质变化如图3所示,乳酸菌不断快速增殖,活菌数由不到1×108CFU/g迅速增长到1.10×1010CFU/g,活菌数扩大100倍以上;酵母由3.2×106CFU/g缓慢增长到5.52×107CFU/g,菌数增殖17倍;pH由初始的6.5不断下降至4.72;乳酸含量不断增加,结束时达6.53%;乙酸产量3.35%;酸度也在发酵过程中持续上升,结束时达到10.65%。
表2发酵乳杆菌F36发酵饲料储存周期内物质变化。
60多天的储存周期内物质变化如表2(图4)所示,乳酸菌和酵母的活菌数不断降低,第9周时乳酸菌活菌数仍有2.8×109CFU/g,酵母活菌数降为1.6×107CFU/g;pH在持续下降,在第5周时降至3.8并在之后基本不变;乳酸产量也略有上升至7.25%,乙酸产量持续维持在3.83%;酸度也略有上升至12.82%。总的来说,在储存期内该发酵饲料理化性质没有较大变化,活菌数有较大幅度降低,但仍保留部分活菌。
实施例5:
发酵乳杆菌F36发酵饲料风味物质分析:
将实施例4中发酵第0d、4d和7d以及储存第28d的饲料样品稀释、离心并过膜处理,再用液相色谱分析有机酸和乙醇含量。色谱柱:Bio-Rad HPX-87H ion-exclusion column,色谱条件:流动相为5mmol H2SO4;柱温40℃;紫外检测器(210nm)和示差折光检测器;流速0.6mL/min;进样量20μL。用GC-MS联用检测样品中的挥发性化合物。结果见表3和表4:
表3气相色谱检测发酵乳杆菌F36发酵饲料发酵及储存中几种酸、醇及糖的含量
注:-表示未检测到;各种物质的单位mg/g。
发酵过程中的主要产物是乳酸和乙酸,分别可达65.3mg/g和33.6mg/g,且储存过程中会有少量增加;同时苹果酸和琥珀酸含量也有部分增加,但是储存过程中会下降,柠檬酸含量减少;酵母活动产生少量的乙醇,含量不断增加;糖含量在发酵过程中会先增加,后降低。
表4 GC-MS检测发酵乳杆菌F36发酵饲料发酵及储存中风味物质含量
注:-表示未检测到;+表示检测到。
GC-MS分析了醇、酮、醛、酸及酯等物质变化,2-ethyl-1-Hexanol、1-Decanol、n-Pentadecanol、n-Nonadecanol-1、2-methyl-1-butanol、(Z)-6,10-dimethyl,5,9-undecadien-2-one、1,1′-oxybis-Octane、Dimethyl ether、2,3-dimethyl undecan、Tetradecane等进40种物质在原料中不存在,由发酵过程中微生物代谢产生;1-Octyltriflfluoroacetate、2-Ethylhexyl salicylate、2,3-dihydro-benzofuran、Pentadecane、4-ethyl-1,3-Benzenediol等8中物质随着发酵时间延长,含量降低至检测不到,被微生物代谢或者转化成其它物质。总之,饲料经微生物发酵会产生更多风味物质,提升饲料的风味,提高其口感和营养价值。
实施例6:
发酵乳杆菌F36发酵饲料在虾饲料上的应用:
虾全价饲料中分别添加实施例4中贮存了30天的发酵饲料10%和20%作为实验组,不添加发酵饲料作为对照组进行小龙虾养殖实验;4月投放100-150尾/斤健康虾苗,每亩虾塘放30斤。两亩作为实验组1,投喂添加10%发酵料的全价料;两亩作为实验组2,投喂添加20%发酵料的全价料,三亩对照组,投喂未添加发酵料的全价料。饲养周期三个月,管理模式相同,每天每亩虾塘的饲料投喂量相同(发酵饲料以干基计算)。养殖结果如表5:
表5复合乳酸菌液体制剂发酵虾全价料养殖实验结果
实验组添加10%发酵饲料小龙虾亩产平均可达206kg,较对照组平均亩产165kg提高24.6%;且实验组小龙虾均重可达40.5g,较对照组均重35g增重14.4%;平均存活率也由78.79%提高到85.76%。添加20%发酵饲料小龙虾亩产平均可达217.5kg,较对照组平均亩产165kg提高31.6%;且实验组小龙虾均重可达42.7g,较对照组均重35g增重22%;平均存活率提高到84.90%。表明小龙虾饲料中添加发酵乳杆菌F36发酵饲料可以提高小龙虾增重以及存活率,且20%的发酵饲料添加量效果优于10%的添加量,养殖效益更好。
实施例7:
发酵乳杆菌F36发酵饲料在生猪养殖中的应用:
在同一舍养殖的生猪中一半做为实验组,另一半做对照组,实验组日粮中添加5%实施例4中贮存了30天的发酵饲料,对照组不添加。分别在保育猪和育肥猪中进行实验,称重后开始实验,实验组与对照组饲喂管理方式相同。保育猪实验组和对照组各150头实验周期30d,育肥猪实验组和对照组各50头实验周期120d,实验结束后称重。
表6保育猪料添加发酵乳杆菌F36发酵饲料实验结果
表6显示保育猪日粮添加10%发酵乳杆菌F36发酵饲料后的实验结果:平均日增重较对照组提高5.87%,实验结束时平均增重0.87公斤,日均采食量提高6.34%,料肉比几乎一样,但是生产能力有明显提高;另外日粮中添加发酵饲料可显著减少保育猪腹泻,增强小猪肠道健康。
表7育肥猪添加发酵乳杆菌F36发酵饲料实验结果
实验组猪较对照组毛色亮丽,更加活跃,猪粪成型度好,臭味少。表7显示育肥猪日粮添加5%发酵乳杆菌F36发酵饲料后的实验结果:实验结束时平均增重2.6公斤,采食量和日增重分别提高1.15%和3.76%,料肉比略微下降,表明添加发酵乳杆菌F36发酵饲料后可促进生猪的采食,提高饲料利用率,显著提高生产能力;另外日粮中添加发酵饲料可显著减少发病率,减少药物成本。
Claims (7)
1.一株分离的发酵乳杆菌(Lactobacillus fermentum ),所述发酵乳杆菌的保藏编号为CCTCC NO:M2020067。
2.权利要求1所述的发酵乳杆菌在制备动物发酵饲料中的应用。
3.根据权利要求2所述的应用,所述的动物发酵饲料为水产动物发酵饲料或畜禽发酵饲料。
4.根据权利要求2所述的应用, 在应用过程包括:
40-60份麸皮、10-30份玉米、15-30份豆粕、3-8份石粉、0.05-1份酸性蛋白酶、0.05-1份中性蛋白酶、0.05-0.2份枯草芽孢杆菌、60份水以及10-20份发酵种子液混合均匀后30-37℃条件下厌氧发酵;
发酵种子液为权利要求1所述的发酵乳杆菌和耐高温酿酒酵母的混合发酵液。
5.根据权利要求4所述的应用,所述的发酵种子液的制备过程包括:
5-10份麸皮、1-2份豆粕加90份水灭菌降温后接种发酵乳杆菌F36种子液,接种量106-107 CFU/ml和耐高温酿酒酵母,接种量104-105 CFU/ml,30-37℃条件下静置发酵即得。
6.权利要求1所述的发酵乳杆菌在制备L-乳酸中的应用。
7.权利要求1所述的发酵乳杆菌在制备淀粉酶中的应用。
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