CN116391792A - 一种非灭菌条件下制备的蛋白发酵饲料 - Google Patents
一种非灭菌条件下制备的蛋白发酵饲料 Download PDFInfo
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Abstract
本发明利用乳酸片球菌SLB‑04和枯草芽孢杆菌YSJL‑30及一株酿酒酵母，以玉米、豆饼(粕)为原料，在非灭菌条件下，采用两段式发酵工艺制备发酵饲料。经单因素试验、正交试验及响应面优化试验，分别获得工艺优化参数。在最佳条件下，发酵饲料氨基酸总量增加3.7个百分点，富含生猪所需十种必需氨基酸，鲜味氨基酸增加幅度明显。高通量测序显示，在种水平上，发酵饲料细菌物种相对丰度中乳酸片球菌和枯草芽孢杆菌为优势菌群，真菌物种相对丰度中酿酒酵母占绝对优势，发酵饲料无沙门氏菌等致病菌感染。本发明为廉价制备生猪发酵饲料提供可行方案，对促进我国生猪饲料的提档升级、高效利用饼粕资源、减少粪便环境污染具有积极意义。
Description
技术领域：
本发明涉及制油副产物饼粕的深度加工利用，以及利用专有菌株乳酸片球菌、枯草芽孢杆菌等在非灭菌条件下联合制备蛋白发酵饲料的工艺及产品。
背景技术：
长期以来，抗生素在饲料中一直起着协助动物机体抵抗病原菌、促进生长的作用，但抗生素的添加致使细菌耐药性和药物残留等问题日益突出，严重威胁人类健康。按照我国农业农村部194号公告规定，自2020年元旦我国饲料中已全面禁止使用抗生素。研究表明发酵饲料被公认是替代抗生素的主要选项。
发酵饲料是利用饲料行业允许的益生菌发酵传统粗饲料以获得更高营养价值的饲料。传统生猪配合饲料中的饼粕含有诸多抗营养因子，如大豆抗原蛋白(大豆球蛋白和β-伴大豆球蛋白)、胰蛋白酶抑制剂、低聚糖、植酸等，降低了生猪饲料蛋白质和矿物质的生物利用率，可引起断奶仔猪肠道的超敏反应，引发腹泻甚至死亡。另外，不被动物消化的蛋白类物质随粪便排到环境，还造成环境污染。
发酵用菌种是发酵饲料质量好坏的关键，发酵饲料常用的微生物菌种包括乳酸菌、芽孢杆菌和酵母菌三类微生物，通过单一或混合菌种发酵，产生抑菌物质抑制致病菌生长繁殖，降解大分子物质，增加菌体蛋白含量，提高饲料的营养价值。我国农业部允许使用的饲用益生菌种有干酪乳杆菌(Lactobacillus casei)、粪链球菌(Streptococcusfaecalis)、乳酸片球菌(pediococcus acidilactici)、纳豆芽孢杆菌(Bacillus natto)、乳链球菌(Streptococcus lactis)、产朊假丝酵母(Candida utilis)、植物乳杆菌(Lactobacillus Plantarum)、屎链球菌(Streptococcus faeciun)、枯草芽孢杆菌(Bacillus subtilis)、嗜酸乳杆菌(Lactobacillus acidophilus)、酿酒酵母(Sacchaeomyces cerevisiae)、沼泽红假单胞菌(Rhodop seudanonas palustris)。例如，乳酸片球菌发酵饲料能够产生有机酸乳酸、抑菌物质细菌素和环二肽，应用到生猪饲喂后，可改善猪的肠道菌群结构、促进猪的肠道发育，改善生猪血清生化指标，提高生猪抗氧化能力，进而促进生猪生长。学者Zheng等人研究发现，枯草芽孢杆菌发酵豆粕，大豆球蛋白、β-伴大豆球蛋白分别降低了86％和70.3％，扫描电子显微镜图像显示，发酵豆粕蛋白质中β构象减少了43.2％，小肽浓度增加。枯草芽孢杆菌发酵豌豆可降低16％的植酸。酿酒酵母发酵羽扇豆，降低植酸含量近80％。芽孢杆菌发酵过程中产生的蛋白酶、淀粉酶、纤维素酶、植酸酶等，可促进饲料原料中蛋白质、淀粉、纤维、脂肪及矿物质的消化吸收率。
发明内容
尽管发酵饲料对生猪生长性能、肠道菌群、免疫调节产生有益作用，但传统发酵饲料制备，按照微生物培养要求需要原料灭菌后接种发酵，尤其是工业化生产发酵饲料需要大型灭菌、冷却设备，这就增加了企业投资和运行成本。而非灭菌式制备发酵饲料又经常出现杂菌污染问题而无法饲喂。为了解决上述问题，本发明利用乳酸片球菌、枯草芽孢杆菌、酿酒酵母的协同作用，采用两段法发酵传统饲料原料，在发酵饲料与原料处理之间寻找平衡点，制备微生物发酵饲料，使之在非灭菌条件下制备的发酵饲料富含多种氨基酸，消除致敏的蛋白抗原，含有多种饲用益生菌并且不含致病菌，具有良好的风味和适口性。
本发明包括以下步骤：
1、一种发酵饲料，其特征在于：以玉米、豆饼(粕)为原料，采用乳酸片球菌SLB-04、枯草芽孢杆菌YSJB-30及酿酒酵母两段式发酵制备蛋白发酵饲料；
所述乳酸片球菌SLB-04，分类命名为乳酸片球菌Pediococcus acidilactici，保存编号为CGMCCNo.24220，保藏日期：2021年12月30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，保藏地址：北京市朝阳区北辰西路1号院3号。所述枯草芽孢杆菌YSJB-30，分类命名为枯草芽孢杆菌Bacillus subtilis，保存编号为CGMCC No.9660，保藏日期：2014年9月15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，保藏地址：北京市朝阳区北辰西路1号院3号。
2、一种发酵饲料，其特征在于：发酵第一阶段以玉米、豆饼(粕)为发酵培养基，发酵培养基无需灭菌处理，接入所述乳酸片球菌SLB-04液体种子液，初始pH 3.5～pH 4.5、水分添加量80％～100％、接种量2％～6％，固态静置发酵48h。发酵第二阶段以第一阶段发酵产物为原料，采用所述枯草芽孢杆菌YSJB-30与酿酒酵母联合发酵，接种量6％，接种比例[枯草芽孢杆菌YSJB-30：酿酒酵母菌](V/V)＝3.8：6.2，发酵温度33.5℃，固态静置发酵48h。
3、一种发酵饲料，其特征在于：发酵饲料富含多种猪必需氨基酸和鲜味氨基酸，并含有益生菌乳酸片球菌SLB-04和枯草芽孢杆菌YSJB-30。
本发明利用乳酸片球菌SLB-04、枯草芽孢杆菌YSJB-30及酿酒酵母的两阶段发酵，在发酵培养基无需灭菌的条件下，通过菌株间的协同作用，避免了饲料中致病菌污染，降解了饲料中的抗营养因子，增加了蛋白质和氨基酸含量，实现了将粗饲料加工成营养丰富的精饲料，利于提高生猪的生长性能和机体免疫力。本发明为廉价制备替代抗生素的生猪发酵饲料提供可行方案，对促进我国生猪饲料的提档升级、高效利用副产物饼粕资源、减少生猪粪便环境污染具有积极意义。
附图说明
图1乳酸片球菌SLB-04的菌落形态和菌体形态
图2乳酸片球菌SLB-04菌株16S rRNA基因PCR产物凝胶电泳检测图
图3乳酸片球菌SLB-04菌株16S rRNA基因序列
图4乳酸片球菌SLB-04菌株16rRNA基因序列与Pediococcus acidilacticistrain 1092 16S ribosomal RNA gene(GenBank:MT573598.1)比对结果
图5本发明发酵饲料制备工艺流程图
图6枯草芽孢杆菌YSJB-30体外抑制致病菌抑菌平板
图7YSBJ-30菌株16S rRNA基因序列与Bacillus subtilis strain B-A 16Sribosomal RNA gene比对结果
图8第一阶段固态发酵水分添加量试验
图9第一阶段固态发酵乳酸菌接种量试验
图10第一阶段固态发酵初始pH值试验
图11第二阶段固态发酵发酵温度单因素试验
图12第二阶段固态发酵接种量单因素试验
图13第二阶段固态发酵接种比例单因素试验
图14发酵体系细菌群落在种水平上的物种相对丰度
图15发酵体系真菌群落在种水平上的物种相对丰度
具体实施方式
具体实施方式一：SLB-04菌株的筛选
无菌条件下，从酸败的果蔬汁中取1mL加入已灭菌、带玻璃珠的20ml生理盐水(9gNaCl/L生理盐水)中，摇匀，取1ml加入9ml无菌生理盐水，混匀，依次梯度稀释至10-6，分别取各梯度样液1ml注入已灭菌的空平皿中，再倾注已灭菌并冷却至约45℃的分离培养基15ml～20ml/个平皿，水平摇匀，凝固后倒置恒温培养箱中，37℃2～3天。挑取菌落周围出现较大透明圈的前10株单菌落穿刺接入灭菌半固体MRS培养基中，37℃培养48h。再将分离菌株接入在MRS液体培养基中37℃培养24h后，按国标1886.173-2016方法测定乳酸含量，如表1，选取产酸高的菌株SLB-04为后续鉴定菌株。
表1产乳酸菌株的筛选
具体实施方式二：菌株SLB-04形态学鉴定
菌株SLB-04在平板培养基上的菌落形态见说明书附图1A，菌株SLB-04可在平板培养基表面生长，菌落小、乳白色，圆形隆起，菌落边缘光滑清晰，无色素分泌，平板培养2天一般菌落直径不超过2mm。
菌株SLB-04光学显微镜下菌体形态如说明书附图1B所示，其扫描电镜菌体形态如说明书附图1C所示。镜下菌体呈球形，直径0.6μm左右，成对出现，罕见单个菌体细胞，常以垂直两个方向分裂呈四联球菌，不形成链状，无芽孢，周围无鞭毛。在斜面培养基中可穿刺生长，兼性厌氧。
具体实施方式三：菌株SLB-04部分生理生化鉴定
将菌株SLB-04部分生理生化试验结果见表2，结果比对《伯杰氏细菌鉴定手册》、《常见细菌系统鉴定手册》中片球菌属特征，其结果一致，进一步判断该菌株可能为片球菌属中的乳酸片球菌。
表2菌株SLB-04与乳酸片球菌部分生理生化试验结果对比表
注：+阳性，-阴性，d表示11％-89％菌株为阳性，＊乳酸片球菌的生理生化试验及结果参照《常见细菌系统鉴定手册》、《伯杰细菌鉴定手册》
具体实施方式四：菌株SLB-04 16SrDNA的分子生物学鉴定
挑取鉴定菌株的斜面菌体接入TaKaRa微生物裂解缓冲液50uL(Code No.9164)中80℃15min变性，离心取上清为PCR模板。采用使用TaKaRa 16S rDNA BacterialIdentification PCR Kit(Code No.RR176)，进行PCR扩增16SrDNA全长片段，SLB-04菌株16S rRNA基因PCR产物凝胶电泳检测图见说明书附图2。
PCR扩增体系50uL：前述变性液1uL，PCR premix 25uL，Forward primer(20pmol/ul)0.5uL，Reverse primer2(20pmol/ul)0.5uL，dd H2O 23uL。PCR扩增条件：94℃5min；94℃1min，55℃1min，72℃1.5min，30cycles；72℃5min。
PCR产物经纯化后，由大连宝生物有限公司完成菌株16SrDNA测序，测序结果获得SEQ ID NO.1，将SEQ ID NO.1与SEQ ID NO.2比对结果见说明书附图4。
结合形态学、生理生化试验、16SrDNA序列分析结果，初步鉴定菌株SLB-04为乳酸片球菌。
具体实施方式五：YSJB-30菌株的筛选
从健康母猪新鲜粪便中分离筛选得到一株枯草芽孢杆菌YSJB-30的方法如下：取新鲜母猪粪便放入无菌容器中，密封，冰盒保存，快速送入实验室进行常规菌种分离。分离平板培养基为添加了脱脂乳的CM培养基。样品无菌条件下，梯度稀释后，将涂布平板于30℃倒置培养1～2天，分别挑取菌落周围出现透明圈的单菌落于CM斜面培养基，共50支，30℃培养1～2天，4℃备用。
无菌条件下，分别将上述50支斜面各一菌环接入灭菌后的液体CM培养基，37℃180rpm摇床培养16h，菌液离心取上清液，进行沙门氏杆菌、单增李斯特菌的抑菌试验，获得抑菌圈大、抑制上述两种致病菌的菌株6株。再分别将6株斜面各一菌环接入液体CM培养基，37℃过夜180rpm培养，测定上清液中蛋白酶活力，取最高酶活菌株，即为实验菌株，命名YSJB-30。
具体实施方式六：提取菌体基因组DNA及16SrDNA的PCR扩增
挑取一菌环斜面菌体接入2ml CM液体培养基，37℃培养16h～18h，取1.5ml活化菌液，12 000r/min离心1min，弃上清液，收集菌体，按照细菌基因组DNA提取试剂盒说明书上的方法提取细菌基因组DNA，-20℃保存备用。0.8％的琼脂糖凝胶检测提取的基因组DNA。
PCR扩增16SrDNA序列：
引物：799F 5’-AACAGGATTAGATACCCTG-3’，1492R 5’-GGTTACCTTGTTACGACTT-3’。
以提取的菌体基因组DNA为模板，进行16SrDNA的PCR扩增。PCR扩增反应体系25μL：10×PCR buffer 2.5uL，dNTP(2.5mM)(TaKaRa)2uL，799F(10pmol/uL)(invitrogen)0.1uL，1492R(10pmol/uL)(invitrogen)0.1uL，Taq聚合酶(5U/uL)(TaKaRa)0.125uL，Templet(提取的DNA)0.5uL，ddH2O to 25uL。
PCR扩增程序：94℃预变性5min。94℃变性1min，52℃退火1min，72℃延伸1min，30个循环，72℃延伸10min。4℃备用。上述PCR产物经1％的琼脂糖凝胶电泳，电压100V，电泳结束，凝胶成像系统观察拍照。
回收16SrDNA PCR扩增产物，送英滩捷基(上海)贸易有限公司北京实验室测序，测序结果获得SEQ ID NO.3。将SEQ ID NO.3与SEQ ID NO.4比对，结合形态学特征，初步鉴定菌株YSJB-30为枯草芽孢杆菌。
具体实施方式七：枯草芽孢杆菌YSJB-30菌株抑菌试验：
取灭菌的液态CM培养基10mL/100mL三角瓶，每瓶接入已活化的枯草芽孢杆菌YSJB-30斜面菌体一菌环，好氧35～36℃培养24h，8000rpm离心1min，取上清液，挑取活化后的单增李斯特菌、沙门氏杆菌菌体各一菌环，分别接入CM液体培养基中37℃培养过夜，梯度稀释适当倍数，涂平板，静置20min后，用打孔器打孔，每孔无菌操作加入上述上清液100ul，静置30min，补加上清液至满孔，37℃培养过夜，观察并测定抑菌圈直径。
具体实施方式八：固态发酵模式的选择
豆饼(粕)粉碎过40目筛，取玉米粉：豆饼(粕)＝3:1，20g/样品，初始水分添加量50％，分别装入塑料袋，以下述三种发酵模式进行固态发酵，接种量均为5％，三种发酵模式如下：
模式1:同时接种乳酸片球菌SLB-04、酿酒酵母菌、枯草芽孢杆菌YSJB-30，静置通气发酵48小时。
模式2:同时接种乳酸片球菌SLB-04、枯草芽孢杆菌YSJB-30密闭发酵24小时，再接种酿酒酵母静置通气发酵24小时。
模式3:接种乳酸片球菌SLB-04密闭发酵24小时，再同时接种酿酒酵母菌和枯草芽孢杆菌YSJB-30静置通气发酵24小时。
测定发酵饲料pH值，结合发酵饲料的色泽、气味及镜检情况进行感官评分，以筛选最佳发酵模式。每个试验三次重复。邀请10名经过培训的感官品评小组人员，对三种不同发酵模式发酵饲料进行感官评价。感官评价参考标准见表3。
表3发酵模式感官评价标准
三种模式发酵感官评价结果如表4所示.
表4发酵模式的感官评价结果。
注：表中同列不同上标字母表示不同样品同一指标差异性显著(P<0.05)。
综合四种感官评价指标，确定C模式为复合益生菌发酵的最佳模式。即先接种乳酸片球菌SLB-04密闭发酵，再同时接种酿酒酵母菌和枯草芽孢杆菌YSJB-30有氧发酵。
具体实施方式八：第一阶段固态发酵条件优化-乳酸片球菌产酸单因素试验
水分添加量试验：取发酵底物20g[玉米粉：豆饼(粕)粉＝3:1]，水分添加量分别为70％、80％、90％、100％、110％，接种量6％，初始pH值4.5，发酵时间48小时，测定发酵饲料乳酸含量，每个试验重复三次，取平均值。结果见图8，选择水分添加量100％为宜。
乳酸菌接种量试验：取发酵底物20g[玉米粉：豆饼(粕)粉＝3:1]，水粉添加量为100％，初始pH值4.5，分别调整接种量2％、4％、6％、8％、10％，发酵时间48小时，测定发酵饲料乳酸含量，每个试验重复三次，取平均值。结果见图9，选择接种量8％为宜。
初始pH值试验：取发酵底物20g[玉米粉：豆饼(粕)粉＝3:1]，水粉添加量为100％，接种量6％，初始pH值分别为3.8、4.1、4.4、4.7、5.0、5.3，发酵48h，测定发酵饲料乳酸含量，每个试验重复三次，取平均值。结果见图10，初始pH值选择pH4.4为宜。
具体实施方式七：第一阶段固态发酵条件优化-乳酸片球菌SLB-04产酸正交试验
如表5所示，根据极差判断，影响乳酸片球菌SLB-04产乳酸量大小的影响因素依次为A(初始pH)>B(水分添加量)>C(接种量)，正交试验优化条件为初始pH4.4、水分添加量100％、接种量6％。
表5正交试验结果
具体实施方式九：第二阶段固态发酵条件优化—单因素试验
第二阶段固态发酵发酵温度单因素试验：按总接种量10％接入混合种子液(枯草芽孢菌：酿酒酵母菌＝1:1)，分别置于25℃、28℃、31℃、34℃静置通气培养48h，取出60℃烘至恒重，测定发酵饲料蛋白质含量，结果如图11。
第二阶段固态发酵接种量单因素试验：以枯草芽孢菌：酿酒酵母菌＝1:1的种子液比例，总接种量分别按6％、8％、10％、12％接入第一阶段发酵饲料，31℃发酵48h，取出60℃烘至恒重，测定发酵饲料蛋白质含量，结果如图12。
第二阶段固态发酵接种比例单因素试验：枯草芽孢杆菌：酿酒酵母菌种子液比例分别取3:7、5:5、7:3、8:2，以总接种量10％接入第一阶段发酵饲料，31℃静置通气48h，取出60℃烘至恒重，测定发酵饲料蛋白质含量，结果如图13。
具体实施方式十：响应面优化第二阶段固态发酵条件
在上述单因素试验基础上，利用Design-Expert 12.0设计响应面试验优化第二阶段固态发酵条件，试验结果如表6所示。
表6响应面试验设计及结果
对表中结果进行多元回归拟合，获得发酵饲料的蛋白含量对接种比例(A)、发酵温度(B)、接种量(C)的二次多项式回归模型为：
Y＝18.22-0.58A-0.51B-0.18C-0.11AB+0.063AC+0.097BC-0.8A2-2.29B2+1.04C2
回归模型及方差分析的结果如表7所示，回归模型差异显著(P＜0.01)，失拟合项差异不显著(P＝0.0689)，表明试验拟合程度良好，设计可靠。回归模型相关系数R2＝0.9023，说明有90.23％的响应值变化与接种比例、发酵温度、接种量有关，预测性良好。由表中F值可知，自变量因子贡献率为A>B>C,即接种比例>发酵温度>接种量，即接种比例对发酵饲料蛋白含量影响最为显著，发酵温度次之，接种量相对影响最小。
表7回归模型及方差分析
注：**表示该项差异极显著(P<0.01)；*表示该项差异显著(P＜0.05)。
经软件优化得到模型的最优工艺参数如表8所示：接种比例为枯草芽孢杆菌：酿酒酵母菌(V/V)＝3.8：6.2，发酵温度33.52℃，接种量6％，此时发酵饲料蛋白含量最高，预测值为19.66％。因发酵温度33.52℃不易操作，故将发酵温度调整为33.5℃。以优化后工艺参数进行3次平行验证试验，测得发酵饲料蛋白含量19.87％，与预测值基本一致，证明模型可靠。与未发酵的饲料原料相比，发酵后的饲料蛋白含量提高了5.21个百分点。经过复合益生菌发酵的饲料从气味上有明显的果香，有回甜，外观色泽明亮。
表8复合益生菌发酵饲料优化结果
具体实施方式十一：
将所制发酵饲料送至黑龙江省农业科学院农产品检测中心检测17种氨基酸，结果如表9。
表9发酵饲料的氨基酸组成
具体实施方式十二：
按照GB13091-2018检测发酵饲料，经黑龙江省农业科学院农产品检测未检测出沙门氏菌。
表10沙门氏菌检测
具体实施方式十三：高通量测序检测发酵体系菌群相对丰度
分别取0h(接种前)、48h(第二阶段接种后)、96h的发酵样品送至天津诺禾致源生物信息科技有限公司，利用PacBio平台进行第三代高通量测序，检测结果见图14和图15。
Claims (4)
1.一种发酵饲料，其特征在于：以玉米、豆饼(粕)为原料，采用乳酸片球菌SLB-04、枯草芽孢杆菌YSJB-30及酿酒酵母两段式发酵制备蛋白发酵饲料；
所述乳酸片球菌SLB-04，分类命名为乳酸片球菌Pediococcus acidilactici，保存编号为CGMCC No.24220，保藏日期：2021年12月30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，保藏地址：北京市朝阳区北辰西路1号院3号；
所述枯草芽孢杆菌YSJB-30，分类命名为枯草芽孢杆菌Bacillus subtilis，保存编号为CGMCC No.9660，保藏日期：2014年9月15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，保藏地址：北京市朝阳区北辰西路1号院3号。
2.一种发酵饲料，其特征在于：
发酵第一阶段以玉米、豆饼(粕)为发酵培养基，无需灭菌处理，采用权利1所述乳酸片球菌SLB-04接种，初始pH 3.5～pH 4.5、水分添加量80％～100％、接种量2％～6％，固态静置发酵48h；
发酵第二阶段以第一阶段发酵产物为原料，采用权利1所述枯草芽孢杆菌YSJB-30与酿酒酵母联合发酵，接种量6％，接种比例[枯草芽孢杆菌YSJB-30：酿酒酵母菌](V/V)＝3.8：6.2，发酵温度33.5℃，固态静置发酵48h。
3.一种发酵饲料，其特征在于：发酵饲料富含多种猪必需氨基酸和鲜味氨基酸，并含有益生菌乳酸片球菌SLB-04和枯草芽孢杆菌YSJB-30。
4.权利要求1-3中任意所述的方法得到的发酵饲料。
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