CN116970513B - 一株植物乳植杆菌sq6及其应用 - Google Patents
一株植物乳植杆菌sq6及其应用 Download PDFInfo
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- CN116970513B CN116970513B CN202310534227.3A CN202310534227A CN116970513B CN 116970513 B CN116970513 B CN 116970513B CN 202310534227 A CN202310534227 A CN 202310534227A CN 116970513 B CN116970513 B CN 116970513B
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- lactobacillus plantarum
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明属于微生物领域,具体涉及一株植物乳植杆菌SQ6及其应用。该植物乳植杆菌SQ6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.27202。本发明筛选出的新菌株植物乳植杆菌SQ6,对诺氟沙星、庆大霉素、多粘菌素B、万古霉素、环丙沙星、卡那霉素、红霉素有耐药性,对多种抗生素,如青霉素、氨苄西林、氧氟沙星等具有敏感性,且对大肠杆菌和金黄色葡萄球菌都有很好的抑制效果;本发明提供的筛选方法简单、高效;筛选的菌株在制备发酵菌剂、进一步制备饲料等方面具有良好的应用前景,将农副产物变废为宝,拓宽了饲料来源,降低了养殖成本,缓解了饲料供应不足的矛盾,有利于畜牧业的发展。
Description
技术领域
本发明属于微生物领域,具体涉及一株植物乳植杆菌SQ6及其应用。
背景技术
近年来,饲料资源的制约逐渐成为世界饲料行业甚至畜牧生产发展的瓶颈。精饲料资源(如玉米、豆粕、鱼粉等)紧缺并且价格较高,而廉价的粗饲料却因无法充分被动物利用而被大量废弃或烧毁,造成资源浪费和环境污染。因此,尝试利用新型饲料原料来代替日渐紧缺的常规饲料原料将会成为未来饲料发展的必然趋势。
粮食深加工的副产物、农副产品的废弃物以及工业有机废水、废渣等含量丰富并且其富含膳食纤维和蛋白质等营养成分。目前对于这些资源的利用还不充分,从而导致这些资源的附加值较低,造成资源的浪费。尤其对于农副产品废弃物的利用存在的问题比较严重,有的直接丢弃,有的进行焚烧。这不仅会造成资源的浪费同时对环境构成破坏。因此,通过微生物发酵的方式来利用这些资源进行饲料生产的研究,不仅可以实现资源的再利用,还能缓解饲料资源紧缺的问题。
目前,除目前使用的部分生理性细菌作为生产菌种外,尚有许多优势菌群未得到开发利用。目前世界许多国家的研发人员正在对饲用微生物进行基础研究,以培育和开发出效果优异的菌株。
发明内容
针对现有技术中存在的问题,本发明提供了一株植物乳植杆菌(Lactiplantibacillus plantarum)SQ6,该菌株从自然发酵大蒜秧中分离获得,产酸性能强。
本发明还提供了上述植物乳植杆菌SQ6在发酵棕榈粕中的应用。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一株植物乳植杆菌(Lactiplantibacillus plantarum)SQ6,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.27202。
本发明还提供了上述植物乳植杆菌SQ6在制备发酵饲料中的应用。
进一步的,所述植物乳植杆菌SQ6发酵的基质为棕榈粕。
本发明还提供了一种含有上述植物乳植杆菌SQ6的菌酶组合制剂,所述菌酶组合制剂为3×106cfu/g植物乳植杆菌+1g/kg复合酶。
本发明还提供了上述菌酶组合制剂在发酵棕榈粕中的应用。
本发明还提供了一种含有上述植物乳植杆菌SQ6的复合菌剂,所述复合菌剂为1.5-3×106cfu/g植物乳植杆菌+1.5-3×106cfu/g戊糖片球菌;所述戊糖片球菌的保藏编号为CGMCC NO.27203。
本发明还提供了一种含有上述复合菌剂的复合菌酶制剂,其特征在于,所述复合菌酶制剂为1.5-3×106cfu/g植物乳植杆菌+1.5-3×106cfu/g戊糖片球菌+1g/kg复合酶。
上述菌酶组合制剂或复合菌酶制剂中所使用的复合酶组成为:
。
本发明还提供了一种上述复合菌剂或复合菌酶制剂在发酵棕榈粕中的应用。
本发明的有益效果为:
(1)本发明筛选出的新菌株植物乳植杆菌SQ6,对诺氟沙星、庆大霉素、多粘菌素B、万古霉素、环丙沙星、卡那霉素、红霉素有耐药性,对多种抗生素,如青霉素、氨苄西林、氧氟沙星等具有敏感性,且对大肠杆菌和金黄色葡萄球菌都有很好的抑制效果;
(2)本发明筛选的菌株,同复合酶进行复配后,制备得到的菌酶组合制剂,具有协同增效作用;同戊糖片球菌复配后,同样具有协同增效作用;本发明提供的菌酶组合制剂、复合菌剂以及复合菌酶制剂,共发酵棕榈粕后,能够大大提高发酵产物中的营养成分含量,并对饲养动物的肠道发育均有较明显的促进效果,从而达到提高饲料的适口性、促进肠道发育的目的;
(3)本发明提供的筛选方法简单、高效;筛选的菌株在制备发酵菌剂、进一步制备饲料等方面具有良好的应用前景,将农副产物变废为宝,拓宽了饲料来源,降低了养殖成本,缓解了饲料供应不足的矛盾,有利于畜牧业的发展。
保藏信息1
保藏时间:2023年04月25日,
保藏单位: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:CGMCC NO.27202,
保藏单位地址: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,
邮政编码:100101
分类命名:植物乳植杆菌Lactiplantibacillus plantarum。
保藏信息2
保藏时间:2023年04月25日,
保藏单位: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:CGMCC NO. 27203,
保藏单位地址: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,
邮政编码:100101
分类命名:戊糖片球菌Pediococcus pentosaceus。
附图说明
图1为实施例1筛选的菌株形貌图;
图2为实施例1筛选的菌株的16SrDNA构建的系统发育树;
图3为实施例1筛选的菌株抑菌效果图;
图4为实施例1筛选的菌株的耐药图;
图5为实施例1筛选的菌株溶血性结果图;
图6为实施例1筛选的菌株的耐酸性对比图;
图7为实施例1筛选的菌株的生长曲线和产酸曲线;
图8为实施例1筛选的菌株发酵过程中pH 的变化速率图;
图9为发酵过程中样品pH结果对比图;
图10为发酵过程中样品活菌数量对比图;
图11为复合菌种及复合酶发酵7 d后棕榈粕的pH结果对比图;
图12为复合菌种及复合酶发酵7 d后棕榈粕中的活菌数对比图;
图13为饲养试验中不同处理组瘤胃微生物群的Chao、Ace、Shannon和Sobs多样性指数。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的解释和说明。
实施例1 菌株筛选
(1)将大蒜收获后,剩余的大蒜秧添加少量的枣粉后置于裹包袋中,用重物压实后进行密封发酵30d左右(采样地点为菏泽郓城);
(2)从自然发酵大蒜秧中分离获得了一株产酸性能强的菌株。采用五点取样法,从自然发酵大蒜秧样品不同位置取样,混合均匀后,称取10g加至90 mL 0.85%无菌生理盐水中,震荡处理,将所得原液进行梯度稀释,吸取10-4、10-5、10-6梯度稀释液上吸取100 μL均匀涂布于含0.3% CaCO3的MRS固体平皿上,于37℃厌氧培养24~48h,挑取产生溶钙圈并具有典型乳酸菌特征的单菌落,连续划线培养2 ~ 3次,直至菌落纯化,经革兰氏染色、形态学观察、分子鉴定将该菌鉴定为植物乳植杆菌,编号为SQ6。
如图1所示:菌体细胞为圆端直杆,单个、成对或呈短链状排列,革兰氏阳性菌。菌落呈圆形,表面光滑,凸起,色白,质地细密,单菌落直径约3 mm左右。
实施例2 16S rDNA分子鉴定
细菌基因组DNA提取参照基因组提取试剂盒方法提取待测菌株基因组DNA。以通用引物(上游引物27F:5’-AGAGTTTGATCCTGGCTCAG-3’,下游引物1492R:5’-GGCTACCTTGTTACGACTT-3’)扩增其16S rDNA片段。PCR 扩增结束后,将电泳结果正确的PCR产物,送至北京擎科生物有限公司(青岛)进行双向测序分析。利用NCBI GenBank 数据库对乳酸菌16S rDNA序列进行BLAST分析,检索与其同源的己知菌种。
PCR扩增体系如表1、表2所示:
表1 16S rDNA扩增反应体系
表2 16S rDNA基因序列的PCR扩增反应条件
将SQ6测序所得16S rDNA序列与GenBank数据库序列进行BLAST比对分析,与Lactobacillus plantarumMT457706.1序列相似度为100%,见表3。采用Neighbor-Joining法与相似度较高序列构建系统发育树,结果见图2。
表3 分离菌SQ6 16S rDNA序列鉴定结果
效果实施例
(一)抑菌性能
将筛选的植物乳植杆菌菌株菌悬液按1%接种于MRS液体培养基中,37℃恒温培养箱孵育24 h后,12,000 r/min离心5 min,取上清液,置于-20℃备用。采用牛津杯法测定,用无菌棉签将活化好的指示菌大肠杆菌(ATCC 25922)和金黄色葡萄球菌(ATCC 25923)菌悬液稀释至1x106cfu/mL,然后分别用无菌棉签浸湿后,沿同一方向涂布LB平板,随后旋转平板60°继续涂布,重复三次,直至涂布均匀。用无菌镊子将灭菌牛津杯平稳置于上述LB固体培养基上,取200 μL制备好的乳酸菌上清液分别加入牛津杯中,置于37℃培养14 h,观察有无抑菌圈产生,并用游标卡尺测量抑菌圈直径大小。
试验结果显示,SQ6菌株对大肠杆菌和金黄色葡萄球菌都有很好的抑制效果(如表4所示),抑菌圈直径分别可达23.4 mm和21.3 mm,如图3所示。
表4 SQ6的对大肠杆菌和金黄色葡萄球菌的抑菌试验结果
(二)抗生素敏感性
将细菌活化后进行梯度稀释,将菌悬液浓度稀释至至1x106cfu/ml,用无菌棉签浸入菌悬液后,沿同一方向涂布整个平板,然后旋转平板60°继续涂布,重复三个角度涂布。采用K-B纸片扩散法进行操作,用无菌镊子取药敏纸片,贴于平板表面,用镊尖轻轻按压纸片,使其贴平。将贴好药敏纸片的平板置于37℃恒温培养箱孵育24~48 h。用游标卡尺量取抑菌圈直径,从不同角度量取三次,取平均值。根据抑菌圈的大小(不同抗生素的抑菌圈大小标准不同),分为敏感(S)、耐药(R)和中介(I)三个标准。
药敏实验按抑菌圈直径大小作为判定敏感度高低的标准。由表5可见,SQ6菌株对本试验所用20种抗生素中诺氟沙星、庆大霉素、多粘菌素B、万古霉素、环丙沙星、卡那霉素、红霉素共7种抗生素都有耐药性,对其余13种抗生素都敏感;图4为筛选的菌株的耐药图。
表5 SQ6菌株抗生素敏感性测定结果
注:“S”代表敏感,“R”代表耐药,“I”代表介于敏感;诺氟沙星:R ≤ 12<I<17 ≤S,氨苄西林:R ≤ 16<I<17 ≤ S,庆大霉素:R ≤ 12<I<15 ≤ S,多粘菌素B:R ≤ 8<I<11≤ S,青霉素:R ≤ 20<I<27 ≤ S,万古霉素:R ≤ 9<I<12 ≤ S,四环素:R ≤ 18<I<22≤S,头孢哌酮:R ≤ 15<I<21≤ S,环丙沙星:R ≤15<I<21≤ S,头孢氨苄:R ≤ 14<I<18 ≤S,多西环素:R ≤ 12<I<16≤ S,头孢拉定:R ≤ 14<I<18≤ S,痢菌净:R ≤ 14<I<17≤S,克林霉素:R ≤ 14<I<21≤ S,卡那霉素:R ≤ 13<I<18≤ S,新霉素:R ≤ 12<I<17≤S,氯霉素:R ≤ 17<I<21≤ S,红霉素:R ≤ 13<I<23≤ S,头孢他啶:R ≤ 14<I<18≤ S
(三)溶血性
挑取适量待测菌株在含5%绵羊血琼脂板上划线培养,37 ℃孵育48 h,观察菌落周边溶血情况。溶血反应检测了β-溶血(菌落周围有清晰的晕)、α-溶血(菌落周围有绿色晕)或γ-溶血(菌落周围无晕,就是不溶血)。从图5中可知,SQ6菌株无溶血性。
(四)耐酸性
分别在不同pH的培养基中培养菌株,测定菌株的最适生长pH和对酸的耐受性。从图6中可以看出:在pH为3.5时,SQ6生长受到抑制;当pH为4时,随着培养基pH的升高菌株的数量不断增加;当pH为5时,SQ6的OD600大于4.0。当pH为原始pH值6.2时,SQ6的OD600大于2.0,因此SQ6在4.0~6.2之间可以生长。
(五)生长曲线与产酸曲线测定
将分离菌株按1%的接种量接入MRS液体培养基中,每隔2 h取样测定其 OD值和pH值,分别绘制生长曲线和产酸曲线。结果如图7所示,菌株在每个时期的生长速度都是不一样的,SQ6在0~4 h内进入细菌生长延迟期,在4 h相继进入对数期。SQ6对数期为8h,结束对数期后进入稳定生长期。
菌株的产酸速率决定了饲料发酵 pH 的变化速率,MRS液体培养基初始pH在6.2左右,结果如图8所示,在细菌培养至0~4 h时培养基内pH值变化不大,此时菌株正处于生长延迟期,生长缓慢,产酸量小;当细菌培养到4 h后,细菌进入对数期后,细菌迅速增殖,开始大量产酸,培养基内pH不断降低;进入稳定期后产酸减少,培养基pH值趋于稳定。
(六)单一菌种与复合酶的发酵试验
将本发明筛选得到的菌株进行发酵试验,具体发酵处理组别如表6所示。
表6
复合酶的酶谱如表7所示:
表7
(1)pH值和活菌数的测定
棕榈粕发酵7d后开袋,五点取样法取发酵棕榈粕20 g,放入40 mL去离子水中,搅拌均匀后用pH计测定样品的pH值。结果如图9所示,添加复合酶的T2处理组比不添加复合酶的T1处理组 pH 有明显的降低,且T2处理组的pH 值最低,为4.31,其他三个处理组 pH 接近。
如图10所示,棕榈粕发酵7d后,T2处理组活菌数量最高,可达7.5×107。其他三个处理组活菌数量明显降低。
(2)营养成分的测定
如下表8所示,经过发酵过后,各处理组的营养成分结果发生了改变。各组的CP含量之间无显著性差异(P>0.05)。T1的DM与T2无显著性差异(P>0.05),显著高于T3和CON(P<0.05)。CON和T3的NDF和ADL显著高于T1和T2组(P<0.05)。T1的ADF显著低于其他组(P<0.05)。CON的Ash与T1和T3之间无显著性差异(P>0.05),显著高于T2(P<0.05)。CON的EE与T2和T3之间无显著性差异(P>0.05),显著高于T1(P<0.05)。T1和T2的NH3-N和WSC显著高于CON和T3(P<0.05)。
表8
如下表9所示,经过发酵过后,各处理组的营养成分结果发生了改变。T2组的LA含量显著高于T1组(P<0.05),T1组的LA含量显著高于T3组(P<0.05),T3组的LA含量显著高于CON组(P<0.05),T2组的AA显著高于T1(P<0.05),T1组的AA显著高于T3和CON(P<0.05)。各组的PA和BA之间无显著性差异(P>0.05)。
表9
(七)复合菌种与复合酶的发酵试验
根据单一菌种及复合酶发酵试验结果,选取植物乳植杆菌和戊糖片球菌搭配复合酶发酵棕榈粕,具体处理分组如表10所示:
表10 复合菌种及复合酶发酵棕榈粕
(1)pH值和活菌数的测定
复合菌及复合酶发酵棕榈粕7 d后开袋,测量其pH值,结果如图11所示,处理组L4高浓度的植物乳植杆菌和戊糖片球菌添加复合酶的pH最低。处理组L2低浓度的植物乳植杆菌和戊糖片球菌添加复合酶的pH也有明显的降低,只添加发酵菌的处理组L1和L3 pH有一定的降低,只添加复合酶处理组pH和对照组pH接近。
由图12可知,复合菌及复合酶发酵棕榈粕7 d后,处理组L4高浓度植物乳植杆菌和戊糖片球菌添加复合酶的活菌数量最高,活菌数可达8.5×107。
(2)营养成分结果指标
由表11可知,经过发酵过后,各处理组的营养成分结果发生了改变。L1组的DM含量最高,显著高于其他处理组(P<0.05),CON和L5组DM含量接近,显著低于其他处理组(P<0.05);CON和L5组的NDF含量接近(P>0.05),显著高于其他处理组(P<0.05);CON、L3和L5组的ADF含量(P>0.05),显著高于其他处理组(P<0.05);粗蛋白含量各处理组之间无显著性差异;L3组和L5组Ash含量接近(P>0.05),显著高于其他处理组;CON、L2和L5组的EE含量接近(P>0.05),显著高于其他处理组(P<0.05);CON、L3和L5组含量接近(P>0.05),显著高于其他处理组(P<0.05);L2、L3和L4的NH3-N含量接近,显著高于其他处理组(P<0.05);L1、L2、L3和L4组的WSC含量接近(P>0.05),显著高于其他处理组(P<0.05)。
表11复合菌种及复合酶发酵7d后棕榈粕的常规营养成分(DM%)
(3)挥发性脂肪酸结果指标
经过发酵后L4组的乳酸含量最高,可达146.03 mmol/L,显著高于其他处理组(P<0.05);L1、L2和L4组的乙酸含量接近(P>0.05),显著高于其他处理组(P<0.05);L2和L4组的丙酸含量接近(P>0.05),显著高于其他处理组(P<0.05);各处理组的丁酸含量接近,无显著性差异(P>0.05),结果如表12所示。
表12复合菌种及复合酶发酵7d后棕榈粕的挥发性脂肪酸影响
(4)饲养试验
处理组别:对照组为牛场原有日粮,处理A组为3%发酵棕榈粕替代部分大豆,处理B为6%发酵棕榈粕替代部分大豆。
①不同处理对肉牛采食量与日增重的影响
每组试验牛的初体重没有显著性差异(P>0.05),试验牛的末体重末体重也没有显著性差异(P>0.05)。从三组试验牛采食量来看,处理B组牛的采食量最高,CON组次之,处理A组最低,但三组牛采食量之间没有显著性差异(P>0.05)。三组试验牛的日增重没有显著性差异(P>0.05),但处理A组的平均日增重最高为1.12kg/d,其次是处理B组的平均日增重为1.10kg/d,CON最低为1.07kg/d。三组试验牛的料重比没有显著性差异(P>0.05),但对照组料重比最高,其次是处理B组,处理A组的料重比最低,结果如表13所示。
表13不同处理对肉牛干物质采食量变化、增重变化及料重比的影响
②不同处理对牛肉品质指标的影响
处理A组和处理B组的初水分含量接近(P>0.05),显著高于CON组(P<0.05);三组试验牛其余肉品质指标之间无显著性差异(P>0.05),结果如表14所示。
表14 不同处理对牛肉品质指标的影响
③ α多样性指数分析
处理A组、处理B组和CON组三组试验牛瘤胃液ACE指数、Chao指数、Shannon指数和Sobs指数均差异显著(P<0.05)。Chao指数、ACE指数和Sobs指数处理A组显著高于CON组(P<0.05),处理A组与处理B组之间无显著性差异(P>0.05),处理B组与CON组之间无显著性差异(P>0.05);Shannon指数处理A组与处理B组之间无显著性差异(P>0.05),都显著高于CON组(P>0.05),如图13所示。
④不同处理对生产成本及经济效益的影响
在不计算养殖过程中人工费、水电费和管理费等情况下,CON组平均每头牛每天纯利润为21.53 元,3%棕榈粕组每天每头牛每天纯利润为23.89 元,6%棕榈粕组每天每头牛每天纯利润为22.45 元,3%棕榈粕组每天每头牛每天纯利润比CON组高2.36 元,6%棕榈粕组每天每头牛每天纯利润比CON组高0.92 元。综上所述,3%棕榈粕组的配方效果较好,成本较低,结果如表15所示。
表15不同处理试验生产成本及经济效益
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Claims (9)
1.一株植物乳植杆菌(Lactiplantibacillus plantarum)SQ6,其特征在于,所述植物乳植杆菌SQ6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.27202。
2.一种如权利要求1所述的植物乳植杆菌SQ6在制备发酵饲料中的应用。
3.根据权利要求2所述的应用,其特征在于,所述的植物乳植杆菌SQ6发酵的基质为棕榈粕。
4.一种含有权利要求1所述的植物乳植杆菌SQ6的菌酶组合制剂,其特征在于,所述菌酶组合制剂为3×106 cfu/g植物乳植杆菌+1g/kg复合酶。
5.一种如权利要求4所述的菌酶组合制剂在发酵棕榈粕中的应用。
6.一种含有如权利要求1所述的植物乳植杆菌SQ6的复合菌剂,其特征在于,所述复合菌剂为1.5-3×106cfu/g植物乳植杆菌+1.5-3×106cfu/g戊糖片球菌;所述戊糖片球菌的保藏编号为CGMCC NO.27203。
7.一种含有权利要求6所述的复合菌剂的复合菌酶制剂,其特征在于,所述复合菌酶制剂为1.5-3×106×106cfu/g植物乳植杆菌+1.5-3×106cfu/g戊糖片球菌+1g/kg复合酶。
8.根据权利要求4所述的菌酶组合制剂或权利要求7所述的复合菌酶制剂,其特征在于,所述复合酶组成为:
。
9.一种如权利要求6所述的复合菌剂或权利要求7所述的复合菌酶制剂在发酵棕榈粕中的应用。
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