CN113774003A - 一种布氏乳杆菌及其在制备低水分发酵饲料中的应用 - Google Patents
一种布氏乳杆菌及其在制备低水分发酵饲料中的应用 Download PDFInfo
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- CN113774003A CN113774003A CN202111330147.3A CN202111330147A CN113774003A CN 113774003 A CN113774003 A CN 113774003A CN 202111330147 A CN202111330147 A CN 202111330147A CN 113774003 A CN113774003 A CN 113774003A
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Abstract
本发明涉及一种布氏乳杆菌及其在制备低水分发酵饲料中的应用,本发明提供了一种布氏乳杆菌TZ‑LB‑017,采用这种布氏乳杆菌制备混合菌液通过两步法制备低水分发酵饲料,即先将部分原料接种菌酶发酵剂,再将其余原料、石粉、磷酸氢钙、氯化钠、氨基酸、预混料等混合后进行二次发酵,通过两步发酵可促进活菌数稳定,更有利于产生有机酸、消化吸收酶等有益物质,同时有利于维生素和矿物质的稳定;本发明制备的低水分发酵饲料可达到至少90天不发霉,且在产酸、抑制有害菌及与其它益生菌的协同作用方面有显著优势;将本发明的低水分发酵饲料与常规饲料进行混合后饲喂,低水分发酵饲料占混合饲料重量比的40‑60%,对于提高畜禽的生产性能有显著促进作用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种布氏乳杆菌及其在制备低水分发酵饲料中的应用。
背景技术
发酵饲料现已经在国外许多国家普遍使用,目前,荷兰有60%以上规模化猪场在使用发酵料,丹麦有30%以上的母猪使用发酵料,法国约有15%的猪场使用发酵料。我国生物发酵饲料的研究与应用也已经成为我国畜牧行业的研究热点。然而,发酵饲料产业在迅猛发展的同时,在实际生产运用中也存在诸多问题,其中,存储以及使用过程中易发霉变质是阻碍发酵饲料广泛应用的重要因素。霉变不仅会导致饲料适口性以及营养价值下降,造成饲料资源浪费,还会对动物的生长性能和健康造成影响,严重时会引起中毒甚至死亡。
菌酶协同生物发酵技术能够有效降解饲料中的大分子及有毒有害物质,产生的菌体蛋白及有益代谢产物,可改善饲料适口性,提升畜禽采食量及饲料利用率。当前,市面上大多数发酵饲料采用一步发酵对单一饲料原料或全价饲料进行发酵处理后,前者直接饲喂,而后者一般与其他组分按比例混合后饲喂。单一原料的发酵饲料已有较多研究,而发酵全价料应用更加方便,但实际生产中存在两个问题,一是直接以全价料为发酵底物发酵,容易导致维生素等营养物质损失;二是常规发酵工艺易发酵不充分,发酵程度浅,水分多在30%以上,高水分不仅在使用过程中搅拌困难,且物料在使用和存储过程中容易产生霉变。三是缺乏高品质的发酵菌种或发酵剂,菌种是饲料发酵工艺技术的关键因素,菌种品质的优劣直接决定发酵饲料的效果与质量。
发明内容
针对现有技术的不足,本发明提供了一种防霉效果好的分步发酵低水分发酵饲料的制备方法,通过两步发酵技术,生产低水分发酵饲料。
本发明提供的一种分步发酵制备低水分发酵饲料的方法,包括如下步骤:
本发明提供了一种布氏乳杆菌TZ-LB-017,分类命名为Lactobacillus buchneri,保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏地址为:北京市朝阳区北辰西路1号院3号,保藏时间为2021年3月23日,保藏编号为CGMCC No.22054。
本发明提供了一种两步法制备低水分发酵饲料的方法,其特征在于,包括如下步骤:
1)制备混合菌液:称取枯草芽孢杆菌菌粉0.01~0.02份、上述布氏乳杆菌TZ-LB-017菌菌粉0.02~0.04份、酿酒酵母菌粉0.005~0.01份 、甘蔗糖蜜0.02~0.04份,加入净化水0.9~0.95份,搅拌充分分散后在25℃~30℃条件下活化3~4h,每隔1h搅拌3~5min,备用;
2)制备复合酶:复合酶中各种酶制剂的质量比为,酸性蛋白酶:酸性甘露聚糖酶:木聚糖酶:纤维素酶:植酸酶:玉米淀粉=(0.2~0.4):(0.05~0.1):(0.1~0.2):(0.1~0.2):(0.05~0.1):(0.2~0.5);
3)制备初混料A:将饲料原料分别除杂、粉碎后,分别称取玉米150.0~350.0份、豆粕120.0~200.0份、玉米胚芽粕30.0份~80.0份、小麦麸50.0~100.0份,将称取的原料在混合机中混合1min~2min,混合均匀后,备用;
4)制备初混料B:分别称取玉米350~550份,石粉4.0~8.0份、磷酸氢钙8.0~12.0份、氯化钠3.0~3.5份、蛋氨酸0 .6~1.0份、70%赖氨酸盐酸盐3.5~6.0份、98%苏氨酸1.0~1.3份、色氨酸0.26~0.34份,混合均匀后,备用;
5)制备预混料:每100重量份预混料中含维生素A 1.30~1.43份,维生素D3 0.53~0.58份,维生素E 10~11份,维生素K3 0.93~1.02份,维生素B2 0.88~0.96份,维生素B6 0.31~0.34份,维生素B12 0.40~0.44 份,烟酰胺4.04~4.44份,泛酸钙2.81~3.09份,生物素0.50~0.55份,五水硫酸铜11.8~12.1份、一水硫酸亚铁31.9~32.2份、一水硫酸锌27.9~28.1份、一水硫酸锰9.3~9.5份、碘酸钙0.05~0 .07份、亚硒酸钠0.02~0.04份,余量的沸石粉;
6)制备发酵初混料A:将2)中制备的复合酶与3)中的制备的初混料A按照1.1~1.5:1000的比例,加入混合机中,混合1~2min,在混合机工作状态下,先加入350份~450份净化水,混合1~2min,再加入1)得到的混合菌液1.0~1.5份,混合均匀后,转移至发酵箱,将发酵箱置于发酵房中28℃~30℃培养48~96h,即可得到发酵初混料A;
7)制备低水分发酵饲料:将6)制备的发酵初混料A、4)制备的初混料B、5)制备的预混料按照质量比350~730:350~560:1.00~1.01的比例进行混合,混合后,装入发酵饲料专用袋中,置于发酵房中28℃~30℃培养48~96h,完成二次发酵,制得低水分发酵饲料。
优选地,步骤1)所述菌粉的有效活菌数如下:枯草芽孢杆菌菌粉1000亿cfu/g,布氏乳杆菌TZ-LB-017菌菌粉为1000亿cfu/g,酿酒酵母菌粉为200亿cfu/g。
优选地,步骤2)中所述各种酶制剂的活性如下:酸性蛋白酶酶活为50000U/g,酸性甘露聚糖酶活为50000U/g,木聚糖酶酶活为400000U/g,纤维素酶活为30000U/g,植酸酶酶活为50000U/g。
本发明同时提供了上述方法制备的低水分发酵饲料在猪饲料中的应用,将低水分发酵饲料与常规饲料进行混合后饲喂,低水分发酵饲料占混合饲料重量比的40-60%。
本发明的有益效果如下:
1、本发明提供了一种从养殖场猪粪便中筛选得到的布氏乳杆菌,该布氏乳杆菌与常规乳酸菌或市售布氏乳杆菌相比,在发酵过程中能够产生具有较高浓度的苯乳酸,与本发明提供的两步法发酵工艺结合,可显著增强发酵饲料的防霉效果,达到至少90天不发霉,且在产酸、抑制有害菌及与其它益生菌的协同作用方面有显著优势。
2、利用了二步法发酵技术,即先将部分原料接种菌酶发酵剂,再将其余原料、石粉、磷酸氢钙、氯化钠、氨基酸、预混料等混合后进行二次发酵,通过两步发酵可促进活菌数稳定,更有利于产生有机酸、消化吸收酶等有益物质,同时有利于维生素和矿物质的稳定。
3、通过二次发酵的配合料整体水分为17.52%~25.29%,低于常规发酵饲料,物料流散性好不易结块,在料线管道中基本无粘附,方便在实际配料中的使用,解决了发酵饲料水分高、生产线中容易粘附、难以推广生产的难题。
4、本发明提供的采用布氏乳杆菌TZ-LB-017及二次发酵工艺的发酵饲料,对于提高畜禽的生产性能有显著促进作用。
附图说明
图1为MRS培养基培养的布氏乳杆菌的菌落形态图片;
图2为实施例2的牛津杯抑菌试验对比图;
图3为实施例4制备的低水分发酵饲料A的苯乳酸含量检测图。
具体实施方式
为了使本技术领域的人员更好地理解本申请方案,下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分的实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本申请保护的范围。
实施例1 筛选布氏乳杆菌
样品来源于本公司试验猪场中断奶仔猪的粪便,将样品在MRS培养基中扩培,扩培后的培养液进行平板涂布,挑取不同形态的乳酸菌菌落,进行平板划线,再挑取单菌落进行二次划线分离,得到7种不同的乳酸菌,然后分别对7种乳酸菌进行产酸性能和苯乳酸含量评价,筛选得到了一株产酸性能较好的乳酸菌为1号乳酸菌。图1为1号乳酸菌(后命名为布氏乳杆菌TZ-LB-017)在MRS培养基中培养24小时的菌落形态。
分离与纯化步骤如下:在无菌条件下称取20份粪便样品20g,分别移至对应的含有180mL无菌蛋白胨培养基中,振荡混匀,静置10min后取上清液备用。 取10mL上清液接种至MRS液体培养基进行增殖培养3次,于37℃恒温培养箱,厌氧培养24h,将增殖后的样品菌悬液用无菌生理盐水梯度稀释到10-7,用倾注平板法各取100μL10-5,10- 6,10- 7浓度的稀释液在含钙MRS培养基平板上涂匀,再覆盖一层含2%琼脂培养基形成厌氧环境,于37℃恒温培养箱中倒置培养48h,挑取溶CaCO3圈较大的菌落多次划线分离纯化直至获得纯种菌株,每处理设3个重复。
产酸性能和苯乳酸含量评价的具体方法为:将7种乳酸菌(编号分别为1-7)作为发酵菌株,进行固体发酵试验,分析不同乳酸菌菌株在发酵料过程中的体系PH变化和苯乳酸含量。取豆粕400份、玉米200份、麸皮400份,进行混合,添加500份水,混合均匀后,平均分为8组,分别接种0.5份生理盐水及上述7种乳酸菌的培养液,装入厌氧发酵袋中,37℃发酵72h后检测其PH值及苯乳酸含量。培养液的制备方法如下:制备无菌MRS肉汤培养基7瓶,分别接种7种乳酸菌,在恒温培养箱中,37℃培养24h,即可得培养液。
可见其中乳酸菌1作为发酵菌株的配合料中,经过高效液相色谱法检测出每克发酵配合料中苯乳酸的含量约为1.51%,远高于其他菌株,将本菌株命名为布氏乳杆菌TZ-LB-017,分类命名为Lactobacillus buchneri,保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏地址为:北京市朝阳区北辰西路1号院3号,保藏时间为2021年3月23日,保藏编号为CGMCC No.22054。
实施例2 验证本株布氏乳杆菌菌株的抑菌效果
采用牛津杯抑菌试验的方法验证布氏乳杆菌(Lactobacillus buchneri)TZ-LB-017抑菌效果,发现该株布氏乳杆菌对金黄色葡萄球菌、大肠菌群都有明显的抑制效果,表明该株布氏乳杆菌对于抑制饲料中常见的有害杂菌革兰氏阴性菌或革兰氏阳性菌都具有显著效果。
实验分别以大肠杆菌、金黄色葡萄球菌作为指示菌,以空白培养基(M)和氨苄青霉素(A)为对照,验证该株布氏乳杆菌的抑菌效果。结果见图2。图中A是抗生素(氨苄霉素)(作为正对照),M是MRS空白培养基(作为对照),LAB是该株布氏乳杆菌培养液。通过观察透周围明圈的大小,判断各种培养液的抑菌效果。结果显示,LAB培养液与正对照组(A)对金黄色葡萄球菌及大肠杆菌均存在明显的抑菌圈,即对金黄色葡萄球菌及大肠杆菌均有抑菌效果,可知该株布氏乳杆菌对于抑制饲料中常见的有害杂菌革兰氏阴性菌或革兰氏阳性菌都具有显著效果。
实施例3 不同布氏乳杆菌制备的发酵饲料防霉性能跟踪
分别以本公司分离得到的布氏乳杆菌TZ-LB-017(实施例1中的乳酸菌1)、市售的布氏乳杆菌40788、市售的布氏乳杆菌KLDS1.0364和市售的常规乳酸菌为发酵菌株,进行固体发酵试验,分析不同布氏乳杆菌菌株在发酵料生产过程中的优势。取豆粕400份、玉米200份、麸皮400份,进行混合,添加500份水,混合均匀后,平均分为5份,分别接种0.5份生理盐水(对照组)、0.5份布氏乳杆菌TZ-LB-017培养液(试验组1)、0.5份常规乳酸菌培养液(试验组2),0.5份布氏乳杆菌40788培养液(试验组3),0.5份布氏乳杆菌KLDS1.0364培养液(试验组4),装入厌氧发酵袋中,37℃发酵72h后取出,敞口放置于恒温恒湿箱中,观察发酵物料的霉变情况。恒温恒湿箱温度设置为30℃,湿度为65%。
乳酸菌和布氏乳杆菌培养液的制备方法如下:制备无菌MRS肉汤培养基4瓶,分别接种常规乳酸菌和3种布氏乳杆菌,在恒温培养箱中,37℃培养24h,即可得常规乳酸菌和3种布氏乳杆菌的培养液。
具体试验结果如下:
试验结果显示,采用本公司分离的布氏乳杆菌TZ-LB-017制备的发酵物料苯乳酸含量显著高于市售其他布氏乳杆菌,防霉效果明显优于常规乳酸菌及其他布氏乳杆菌,与市售布氏乳杆菌相比,可以延长发酵物料保质期达30天之久。
实施例4 制备低水分发酵饲料A
1、混合菌种制备:称取枯草芽孢杆菌菌粉0.01份、布氏乳杆菌TZ-LB-017菌粉0.02份、酿酒酵母菌粉0.005份 、甘蔗糖蜜0.02份,加入净化水0.945份,搅拌充分分散后,将制备好的混合菌种在25℃~30℃条件下,活化3.5 h,每隔1h搅拌4 min,备用;其中菌粉有效活菌数如下,枯草芽孢杆菌菌粉有效活菌数为1000亿cfu/g,布氏乳杆菌TZ-LB-017菌粉有效活菌数为1000亿cfu/g,酿酒酵母菌粉有效活菌数为200亿cfu/g。
2、复合酶的制备:称取酸性蛋白酶粉末0.2份、酸性甘露聚糖酶粉末0.05份、木聚糖酶粉末0.1份、纤维素酶粉末0.1份、植酸酶粉末0.05份,玉米淀粉0.5份,混匀,即为1份复合酶,备用;其中,酸性蛋白酶酶活为50000U/g,酸性甘露聚糖酶活为50000U/g,木聚糖酶酶活为400000U/g,纤维素酶活为30000U/g,植酸酶酶活为50000U/g。
3、初混料A的制备:将饲料原料分别除杂、粉碎后,分别称取玉米150份、豆粕150.0份、玉米胚芽粕40.0份、小麦麸80.0份,将称取的原料在混合机中混合1min,混合均匀后,备用。
4、初混料B的制备:分别称取玉米 550份,石粉 8.0份、磷酸氢钙 12.0份、氯化钠3.0份、蛋氨酸 1.0份、70%赖氨酸盐酸盐 6.0份、98%苏氨酸 1.3份、色氨酸0.28份,混合均匀后,备用。
5、预混料的制备:每100份预混料中含维生素A 1.35份,维生素D3 0.55份,维生素E 10份,维生素K3 0.98份,维生素B2 0.92份,维生素B6 0.32份,维生素B12 0.42 份,烟酰胺4.24份,泛酸钙2.89份,生物素0.53份,五水硫酸铜11.9份、一水硫酸亚铁32份、一水硫酸锌28.1份、一水硫酸锰9.5份、碘酸钙0.05份、亚硒酸钠0.02份,余量的沸石粉。
6、将(2)中制备的复合酶与(3)中的制备的初混料A按照1:1000的比例,加入混合机中,混合1min,在混合机工作状态下,先加入350份净化水,混合1min,再加入混合菌种1份,混合均匀后,转移至发酵箱,将发酵箱置于发酵房中培养72h,即可得到发酵初混料A。其中,发酵房温度控制在28℃~30℃之间。
7、将(6)中制备的全部发酵初混料A、(4)中制备的初混料B、(5)中的1份预混料按照1:1:0.04进行混合,混合后,装入发酵饲料专用袋中,置于发酵房中培养72h,完成二次发酵,即可得到菌酶协同的低水分发酵饲料。其中,发酵房温度控制在28℃~30℃之间。
本实施例获得的低水分发酵饲料水分含量为19.98%,低于常规发酵饲料30%以上的水分含量,生产过程中物料流散性好不易结块,在料线管道中基本无粘附。
采用高效液相色谱法,检测本实施例获得的低水分发酵饲料中有机酸的种类,如图3所示,检测结果检测每g低水分发酵饲料中含有0.015g苯乳酸。
实施例5 制备低水分发酵饲料B
1、混合菌种制备:称取枯草芽孢杆菌菌粉0.015份、布氏乳杆菌TZ-LB-017菌粉0.04份、酿酒酵母菌粉0.01份 、甘蔗糖蜜0.03份,加入净化水0.915份,搅拌充分分散后,将制备好的混合菌种在25℃条件下,活化3h,每隔1h搅拌3~5min,备用;其中菌粉有效活菌数如下,枯草芽孢杆菌菌粉有效活菌数为1000亿cfu/g,布氏乳杆菌TZ-LB-017菌菌粉有效活菌数为1000亿cfu/g,酿酒酵母菌粉有效活菌数为200亿cfu/g。
2、复合酶的制备:称取酸性蛋白酶粉末0.3份、酸性甘露聚糖酶粉末0.1份、木聚糖酶粉末0.1份、纤维素酶粉末0.1份、植酸酶粉末0.1份、玉米淀粉0.3份,混合均匀,即为1份复合酶,备用;其中,酸性蛋白酶酶活为50000U/g,酸性甘露聚糖酶活为50000U/g,木聚糖酶酶活为400000U/g,纤维素酶活为30000U/g,植酸酶酶活为50000U/g。
3、初混料A的制备:将饲料原料分别除杂、粉碎后,分别称取玉米150份、豆粕150.0份、玉米胚芽粕40.0份、小麦麸80.0份,将称取的原料在混合机中混合1min,混合均匀后,备用。
4、初混料B的制备:分别称取玉米 550份,石粉 8.0份、磷酸氢钙 12.0份、氯化钠3.0份、蛋氨酸 1.0份、70%赖氨酸盐酸盐 6.0份、98%苏氨酸 1.3份、色氨酸0.28份,混合均匀后,备用。
5、预混料的制备:每100份预混料中含维生素A 1.35份,维生素D3 0.55份,维生素E 10份,维生素K3 0.98份,维生素B2 0.92份,维生素B6 0.32份,维生素B12 0.42 份,烟酰胺4.24份,泛酸钙2.89份,生物素0.53份,五水硫酸铜11.9份、一水硫酸亚铁32份、一水硫酸锌28.1份、一水硫酸锰9.5份、碘酸钙0.05份、亚硒酸钠0.02份,余量的沸石粉。
6、将(2)中制备的复合酶与(3)中的制备的初混料A按照1:1000的比例,加入混合机中,混合1min,在混合机工作状态下,先加入350份净化水,混合1min,再加入混合菌种1份,混合均匀后,转移至发酵箱,将发酵箱置于发酵房中培养72h,即可得到发酵初混料A。其中,发酵房温度控制在28℃~30℃之间。
7、取(4)中制备的全部初混料B、(5)中的1份预混料、(6)中制备的全部发酵初混料A进行混合,混合后,装入发酵饲料专用袋中,置于发酵房中培养72h,完成二次发酵,即可得到菌酶协同的低水分发酵饲料。其中,发酵房温度控制在28℃~30℃之间。
本实施例获得的低水分发酵饲料水分含量为19.63%,低于常规发酵饲料30%以上的水分含量,生产过程中物料流散性好不易结块,在料线管道中基本无粘附。
实施例6 制备低水分发酵饲料C
1、混合菌种制备:称取枯草芽孢杆菌菌粉0.02份、布氏乳杆菌TZ-LB-017菌粉0.03份、酿酒酵母菌粉0.01份 、甘蔗糖蜜0.03份,加入净化水0.9份,搅拌充分分散后,将制备好的混合菌种在25℃~30℃条件下,活化4h,每隔1h搅拌5min,备用;其中菌粉有效活菌数如下,枯草芽孢杆菌菌粉有效活菌数为1000亿cfu/g,布氏乳杆菌TZ-LB-017菌粉有效活菌数为1000亿cfu/g,酿酒酵母菌粉有效活菌数为200亿cfu/g。
2、复合酶的制备:称取酸性蛋白酶粉末0.4份、酸性甘露聚糖酶粉末0.1份、木聚糖酶粉末0.2份、纤维素酶粉末0.2份、植酸酶粉末0.1份,玉米淀粉0.2份,混匀,即为1份复合酶,备用;其中,酸性蛋白酶酶活为50000U/g,酸性甘露聚糖酶活为50000U/g,木聚糖酶酶活为400000U/g,纤维素酶活为30000U/g,植酸酶酶活为50000U/g。
3、初混料A的制备:将饲料原料分别除杂、粉碎后,分别称取玉米150份、豆粕150.0份、玉米胚芽粕40.0份、小麦麸80.0份,将称取的原料在混合机中混合1min,混合均匀后,备用。
4、初混料B的制备:分别称取玉米 550份,石粉 8.0份、磷酸氢钙 12.0份、氯化钠3.0份、蛋氨酸 1.0份、70%赖氨酸盐酸盐 6.0份、98%苏氨酸 1.3份、色氨酸0.28份,混合均匀后,备用。
5、预混料的制备:每100份预混料中含维生素A 1.35份,维生素D3 0.55份,维生素E 10份,维生素K3 0.98份,维生素B2 0.92份,维生素B6 0.32份,维生素B12 0.42 份,烟酰胺4.24份,泛酸钙2.89份,生物素0.53份,五水硫酸铜11.9份、一水硫酸亚铁32份、一水硫酸锌28.1份、一水硫酸锰9.5份、碘酸钙0.05份、亚硒酸钠0.02份,余量的沸石粉。
6、将(2)中制备的复合酶与(3)中的制备的初混料A按照1:1000的比例,加入混合机中,混合1min,在混合机工作状态下,先加入350份净化水,混合1min,再加入混合菌种1份,混合均匀后,转移至发酵箱,将发酵箱置于发酵房中培养72h,即可得到发酵初混料A。其中,发酵房温度控制在28℃~30℃之间。
7、取(4)中制备的全部初混料B、(5)中的1份预混料、(6)中制备的全部发酵初混料A进行混合,混合后,装入发酵饲料专用袋中,置于发酵房中培养72h,完成二次发酵,即可得到菌酶协同的低水分发酵饲料。其中,发酵房温度控制在28℃~30℃之间。
本实施例获得的低水分发酵饲料水分含量为19.46%,低于常规发酵饲料30%以上的水分含量,生产过程中物料流散性好不易结块,在料线管道中基本无粘附。
实施例7一步法发酵饲料料的制备
1、混合菌种制备:称取枯草芽孢杆菌菌粉0.02份、布氏乳杆菌TZ-LB-017菌粉0.02份、酿酒酵母菌粉0.01份 、甘蔗糖蜜0.03份,加入净化水0.9份,搅拌充分分散后,将制备好的混合菌种在25℃~30℃条件下,活化4h,每隔1h搅拌5min,备用;其中菌粉有效活菌数如下,枯草芽孢杆菌菌粉有效活菌数为1000亿cfu/g,布氏乳杆菌TZ-LB-017菌粉有效活菌数为1000亿cfu/g,酿酒酵母菌粉有效活菌数为200亿cfu/g。
2、复合酶的制备:称取酸性蛋白酶粉末0.4份、酸性甘露聚糖酶粉末0.1份、木聚糖酶粉末0.2份、纤维素酶粉末0.2份、植酸酶粉末0.1份,玉米淀粉0.2份,混匀,即为1份复合酶,备用;其中,酸性蛋白酶酶活为50000U/g,酸性甘露聚糖酶活为50000U/g,木聚糖酶酶活为400000U/g,纤维素酶活为30000U/g,植酸酶酶活为50000U/g。
3、配合料的制备:将饲料原料分别除杂、粉碎后,分别称取玉米700份、豆粕150.0份、玉米胚芽粕40.0份、小麦麸80.0份、石粉 8.0份、磷酸氢钙 12.0份、氯化钠 3.0份、蛋氨酸 1.0份、70%赖氨酸盐酸盐 6.0份、98%苏氨酸 1.3份、色氨酸0.28份,1份预混料、装入混合机,混合3-5min,混合均匀后,备用。其中,每100份预混料中含维生素A 1.35份,维生素D30.55份,维生素E 10份,维生素K3 0.98份,维生素B2 0.92份,维生素B6 0.32份,维生素B120.42 份,烟酰胺4.24份,泛酸钙2.89份,生物素0.53份,五水硫酸铜11.9份、一水硫酸亚铁32份、一水硫酸锌28.1份、一水硫酸锰9.5份、碘酸钙0.05份、亚硒酸钠0.02份,余量的沸石粉。
4、将(2)中制备的复合酶与(3)中制备的配合料按照1:1000的比例,加入混合机中,混合1min,在混合机工作状态下,先加入350份净化水,混合1min,再加入(1)中制备的混合菌种1份,混合均匀后,转移至发酵箱,将发酵箱置于发酵房中培养72h,即可得到常规发酵饲料。其中,发酵房温度控制在28℃~30℃之间。
本实施例获得的低水分发酵饲料水分含量为19.48%,低于常规发酵饲料30%以上的水分含量,生产过程中物料流散性好不易结块,在料线管道中基本无粘附。
从实施例4-7可见,采用布氏乳杆菌TZ-LB-017配合两步法发酵制备的低水分发酵饲料的水分含量约为17.52%~25.29%,低于常规发酵饲料。本发明的方法制备的低水分发酵饲料流散性好不易结块,在料线管道中基本无粘附,不增加工人的额外劳动,对生产线友好。
实施例8 低水分发酵饲料品质检测
分别对实施例4-7中的发酵饲料与常规未发酵饲料的品质进行检测,结果如下表:
对检测数据分析可知,在都采用本发明提供的布氏乳杆菌TZ-LB-017菌粉的情况下,实施例4-7获得的低水分发酵饲料中,都能检测出苯乳酸,且采用两步法发酵的实施例4-6的苯乳酸含量显著高于采用一步法发酵的实施例7;实施例4-7获得的发酵饲料的益生菌都大量繁殖,其中采用两步法发酵的实施例4-6,总酸含量显著高于实施例7的一步法发酵饲料和对照组的未发酵饲料,大豆球蛋白和大豆β~伴球蛋白这两种大豆抗原蛋白及呕吐毒素均显著低于实施例7和对照组,黄曲霉毒素均未测出。说明采用本发明的布氏乳杆菌TZ-LB-017,同时采用本发明提供的两步法进行发酵的发酵饲料,能够有效提升配合饲料中的粗蛋白、酸溶蛋白、总酸,有效降低饲料中的大豆抗原蛋白及霉菌毒素的含量,同时,在发酵过程中,益生菌大量繁殖,伴随着多种有益代谢产物的产生,对于提高饲喂动物的生产性能有显著影响。
实施例9 低水分发酵饲料稳定性检测
分别于0天、30天、60天、90天,检测实施例4两步法制备的样品A的微生物数量、pH、及感官指标,并与实施例7一步法制备的发酵饲料的感官指标进行对比,检测结果如下:
试验结果显示,采用本发明的布氏乳杆菌TZ-LB-017,同时采用本发明提供的两步法进行发酵的发酵饲料,在90天(三个月)的保存过程中,微生物的数量仍在缓慢的增长,而pH在缓慢地降低,说明物料中微生物仍在缓慢地代谢,其次物料气味酸香,未检出霉菌,说明采用本发明技术方案制备的发酵饲料保质期至少达到90天;而采用本发明的布氏乳杆菌TZ-LB-017,同时采用一步法的发酵饲料,在60天出现局部小霉斑现象,到90天时,已完全呈现霉变现象,这个试验与实施例3的仅用玉米豆粕麸皮的试验结果也相互印证,说明本发明的布氏乳杆菌TZ-LB-017的确能延长饲料原料发酵的保质期达30天左右,但用一步法发酵并不能很好的发挥布氏乳杆菌TZ-LB-017抗霉变的潜力。也说明,在采用本发明的布氏乳杆菌TZ-LB-017的同时采用本发明的两步法发酵步骤,能进一步延长发酵饲料的保质期、发酵饲料的稳定性更好。
实施例10 动物饲喂对比试验 实施例4~6的动物试验效果
选取河北遵化某猪场进行饲喂试验验证,选取40头75kg左右的生长肥育猪,随机分为4栏,每栏10头,对照组饲喂未发酵的常规饲料,试验组1~3分别对应饲喂实施例4~6中制备的低水分发酵饲料与未发酵的常规饲料的混合料,其中低水分发酵饲料占50%,未发酵的常规饲料占50%;试验天数为30天,常规饲喂方式和饮水,对照组合试验组每天的饲喂量和饲喂次数相同。试验期间观察对照组和试验组的猪只变化情况,结果如下表。
试验结果显示,与对照组相比,在生长肥育猪日粮中添加50%的低水分发酵饲料,提高了日增重,降低料肉比,提高投入产出比。通过该组试验也证明了本发明的低水分发酵饲料在基础日粮中大比例添加,能够有效地促进生长肥育猪的生产性能,提高饲料转化率。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (5)
1.一种布氏乳杆菌(Lactobacillus buchneri)TZ-LB-017,保藏编号为CGMCCNo.22054。
2.一种两步法制备低水分发酵饲料的方法,其特征在于,包括如下步骤:
1)制备混合菌液:称取枯草芽孢杆菌菌粉0.01~0.02份、权利要求1所述布氏乳杆菌TZ-LB-017菌菌粉0.02~0.04份、酿酒酵母菌粉0.005~0.01份 、甘蔗糖蜜0.02~0.04份,加入净化水0.9~0.95份,搅拌充分分散后在25℃~30℃条件下活化3~4h,每隔1h搅拌3~5min,备用;
2)制备复合酶:复合酶中各种酶制剂的质量比为,酸性蛋白酶:酸性甘露聚糖酶:木聚糖酶:纤维素酶:植酸酶:玉米淀粉=(0.2~0.4):(0.05~0.1):(0.1~0.2):(0.1~0.2):(0.05~0.1):(0.2~0.5);
3)制备初混料A:将饲料原料分别除杂、粉碎后,分别称取玉米150.0~350.0份、豆粕120.0~200.0份、玉米胚芽粕30.0份~80.0份、小麦麸50.0~100.0份,将称取的原料在混合机中混合1min~2min,混合均匀后,备用;
4)制备初混料B:分别称取玉米350~550份,石粉4.0~8.0份、磷酸氢钙8.0~12.0份、氯化钠3.0~3.5份、蛋氨酸0 .6~1.0份、70%赖氨酸盐酸盐3.5~6.0份、98%苏氨酸1.0~1.3份、色氨酸0.26~0.34份,混合均匀后,备用;
5)制备预混料:每100重量份预混料中含维生素A 1.30~1.43份,维生素D3 0.53~0.58份,维生素E 10~11份,维生素K3 0.93~1.02份,维生素B2 0.88~0.96份,维生素B6 0.31~0.34份,维生素B12 0.40~0.44 份,烟酰胺4.04~4.44份,泛酸钙2.81~3.09份,生物素0.50~0.55份,五水硫酸铜11.8~12.1份、一水硫酸亚铁31.9~32.2份、一水硫酸锌27.9~28.1份、一水硫酸锰9.3~9.5份、碘酸钙0.05~0 .07份、亚硒酸钠0.02~0.04份,余量的沸石粉;
6)制备发酵初混料A:将2)中制备的复合酶与3)中的制备的初混料A按照1.1~1.5:1000的比例,加入混合机中,混合1~2min,在混合机工作状态下,先加入350份~450份净化水,混合1~2min,再加入1)得到的混合菌液1.0~1.5份,混合均匀后,转移至发酵箱,将发酵箱置于发酵房中28℃~30℃培养48~96h,即可得到发酵初混料A;
7)制备低水分发酵饲料:将6)制备的发酵初混料A、4)制备的初混料B、5)制备的预混料按照质量比350~730:350~560:1.00~1.01的比例进行混合,混合后,装入发酵饲料专用袋中,置于发酵房中28℃~30℃培养48~96h,完成二次发酵,制得低水分发酵饲料。
3.根据权利要求2所述两步法制备低水分发酵饲料的方法,其特征在于,步骤1)所述菌粉的有效活菌数如下:枯草芽孢杆菌菌粉1000亿cfu/g,布氏乳杆菌TZ-LB-017菌菌粉为1000亿cfu/g,酿酒酵母菌菌粉为200亿cfu/g。
4.根据权利要求2所述两步法制备低水分发酵饲料的方法,其特征在于,步骤2)中所述各种酶制剂的活性如下:酸性蛋白酶酶活为50000U/g,酸性甘露聚糖酶活为50000U/g,木聚糖酶酶活为400000U/g,纤维素酶活为30000U/g,植酸酶酶活为50000U/g。
5.如权利要求2所述方法制备的低水分发酵饲料在猪饲料中的应用,其特征在于,将低水分发酵饲料与常规饲料进行混合后饲喂,低水分发酵饲料占混合饲料重量比的40-60%。
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