CN115316496A - 一种利用废弃羽毛制备高蛋白益生动物饲料的方法 - Google Patents
一种利用废弃羽毛制备高蛋白益生动物饲料的方法 Download PDFInfo
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Abstract
本发明公开了一种利用废弃羽毛制备高蛋白益生动物饲料的方法,是膨化的羽毛先经乳酸菌发酵,发酵液灭活后接种枯草芽孢杆菌种子液,通过补加葡萄糖和膨化羽毛进行高密度发酵,获得益生菌体及其降解羽毛的混合物;向混合物添加甘蔗渣、麦麸、淀粉,造粒后制得高蛋白益生动物饲料。本发明联合枯草芽孢杆菌与乳酸菌降解废弃羽毛制备的动物饲料含有较高的粗蛋白(37.2%)、可溶性蛋白(27.1g/L)和活性益生菌数(2.5×108cfu/g),具有改善风味与促进肠道建立微生态稳态、提高免疫力与抗病能力以及可替代抗生素的潜力。该方法无需除菌、成本低、易于推广。本发明将废弃羽毛高值化利用且提供益生的高蛋白饲料资源,促进畜禽养殖业的持续健康发展。
Description
技术领域
本发明属于饲料技术领域,具体涉及了一种利用废弃羽毛制备高蛋白益生动物饲料的方法。
背景技术
养殖业的集约化和规模化发展增大了禽畜动物对蛋白饲料的需求。我国蛋白饲料的缺口较大且其原料的进口依存度接近80%,这严重制约了我国饲料工业和养殖业的健康发展。废弃羽毛是畜禽养殖业的主要副产物且在我国的年产量达数百万吨,其粗蛋白含量在90%左右;与昂贵的豆粕及其替代物相比,它是一种天然的廉价优质蛋白质资源。将废弃羽毛用作蛋白饲料的蛋白源既能解决其对环境和人类健康带来的危害,又可缓解我国蛋白饲料资源短缺的问题,具有重要的工业应用价值。
羽毛结构富含高度交联的二硫键是限制其高效降解和利用的瓶颈问题。采用高温膨化法、蒸煮法和酶解法将废弃羽毛转化成羽毛粉是目前废弃羽毛的主要处理方式。然而,畜禽动物对羽毛粉的消化率较低。由于羽毛结构还含有其它共价键如氢键,需要多种酶的协同作用来降解羽毛,但酶的制备成本高。目前,利用微生物菌株及其产生以角蛋白酶为主的发酵液降解羽毛已备受关注。
乳酸菌通过定植占位和抑制病害菌生长来调节胃肠道菌群并维持其微生态平衡,进而改善胃肠道功能;通过代谢有机生物质产生的乳酸等有机酸可进一步抑制致病菌繁殖和促进机体生长。因此,乳酸菌可有效提高免疫力和防治胃肠道疾病(如腹泻)。
发明内容
本发明的目的是提供一种利用废弃羽毛制备高蛋白益生动物饲料的方法。该方法联合芽孢杆菌与乳酸菌降解废弃羽毛并制备高蛋白益生动物饲料,饲料的可溶性蛋白含量达到27.1g/l、活性益生菌浓度达到2.5×108cfu/g。
本发明的利用废弃羽毛制备高蛋白益生动物饲料的方法,是膨化的羽毛先经乳酸菌发酵,乳酸菌发酵液灭活后接种枯草芽孢杆菌种子液,通过分批补加葡萄糖和膨化的羽毛进行生物发酵和降解,获得降解羽毛的混合物;
在混合物中添加甘蔗渣、麦麸、淀粉,复配混匀后造粒,制得高蛋白益生动物饲料。
优选,所述的乳酸菌是发酵乳杆菌、副干酪乳杆菌或植物乳杆菌。
优选,所述的枯草芽孢杆菌是枯草芽孢杆菌(Bacillus subtilis)HC2。
优选,所述的废弃羽毛为鸡毛、鸭毛或鹅毛。
优选,利用废弃羽毛制备高蛋白益生动物饲料的方法,具体为:
1)将乳酸菌接种于MRS培养基,在36℃培养16~20h,以体积比2%接种量接种至发酵培养基,在36℃培养2~3天,结束后在60℃热处理2h;
所述的发酵培养基:按质量分数计,包括0.01~0.09%NaCl、0.02~0.1%KH2PO4、0.01~0.09%K2HPO4、0.01~0.09%MgSO4,6~8%膨化的羽毛,余量为水,pH为8.5~9.5,121℃灭菌20min;
2)将枯草芽孢杆菌接种于液体LB培养基,在37℃和160~240r/min条件下培养10~18h,制得种子液;
3)将种子液以体积比2~10%的接种量接种至步骤1)热处理后的发酵培养液中,在37℃和160~240r/min条件下通过分批补加葡萄糖和膨化的羽毛,其中葡萄糖是按发酵液体积的0.5%(质量体积比,w/v)补加,膨化的羽毛是按发酵液体积的5~8%(质量体积比,w/v)补加,进行高密度发酵和降解羽毛2~5天,获得降解羽毛的混合物;其中,碱性角蛋白酶的活力在1000U/ml以上。
4)将步骤3)混合物与混合物质量的8~12%甘蔗渣、混合物质量的9~15%麦麸、混合物质量的8~12%淀粉复配混匀,在220~240r/min条件下造粒,制得高蛋白益生动物饲料。
优选,所述的发酵培养基:按质量分数计,0.06%NaCl、0.05%KH2PO4、0.02%K2HPO4、0.06%MgSO4,6%膨化的羽毛,pH为9.0,121℃灭菌20min。
获得高蛋白益生动物饲料的粗蛋白含量为37.2%,可溶性蛋白含量为27.1g/L,粗纤维含量为6.3%;发酵液中益生菌活菌数为5.12×108cfu/ml,饲料中的益生菌活菌数则达到2.5×108cfu/g。制备的高蛋白饲料仍保持较高的活性益生菌数,高于Q/HBMSY 001-2019企业标准的复合益生菌发酵饲料的益生菌数量,具有益生功能和替代抗生素的巨大潜力。
乳酸菌已作为饲料添加剂替代抗生素用于动物饲料,而将其用于降解废弃羽毛并制备高蛋白益生动物饲料尚未见报道。同时,乳酸菌产生的乳酸对羽毛和羽毛粉具有一定的软化作用,且具有降低原料不良风味和提高饲料适口性的功能。因此,联合羽毛降解菌株枯草芽孢杆菌与乳酸菌降解废弃羽毛并制备高蛋白益生动物饲料对废弃羽毛的高值化利用和提供益生蛋白饲料资源及养殖业的持续健康发展具有重要的推动作用。
与现有技术相比,本发明具有以下优点:
1、枯草芽孢杆菌与乳酸菌的联合使用显著增强了碱性角蛋白活力。
2、直接采用益生菌(枯草芽孢杆菌和乳酸菌)降解废弃羽毛制备饲料,而非以添加剂形式用于饲料。
3、饲料含有更高浓度的粗蛋白(~37.2%)和可溶性蛋白(~27.1g/l),活性益生菌数量达到2.5×108cfu/g,具备高蛋白益生动物饲料特征。
4、该饲料制备过程操作简单、无需离心除菌或灭菌、成本低,易于推广。
Bacillus subtilis HC2,其于2022年5月12日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广州市先烈中路100号大院59号楼5楼,邮编:510070,保藏号GDMCC No:62466。
附图说明
图1是发酵液中角蛋白酶活力的比较。
图2是制备的高蛋白益生动物饲料。
具体实施方式
以下实施例是对本发明进一步说明,而不是对本发明的限制。
以下实施例使用的枯草芽孢杆菌(Bacillus subtilis)是本发明人从土壤环境样品中分离得到的枯草芽孢杆菌(Bacillus subtilis)HC2,其于2022年5月12日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广州市先烈中路100号大院59号楼5楼,邮编:510070,保藏号GDMCC No:62466。
以下实施例使用的发酵乳杆菌是常规发酵乳杆菌,可以从各大保藏中心买到发酵乳杆菌。
实施例1高蛋白益生动物饲料的制备
利用双螺杆挤压膨化机在180r/min、160℃和0.4MPa条件下膨化废弃鸡羽毛(含水量约18%)获得羽毛粉。将发酵乳杆菌接种至MRS培养基,在36℃培养18h作为种子液;以体积比2%接种量接种至发酵培养基,在36℃培养2天后进行热处理(60℃左右)2h。同时,将枯草芽孢杆菌HC2挑至液体LB培养基在37℃和180r/min培养18h作为种子液,以体积比6%接种量转接至上述热处理后的发酵培养液(500ml)中,在37℃和180r/min条件下通过分批补加葡萄糖和羽毛粉,其中葡萄糖是按发酵液体积的0.5%(质量体积比,w/v)补加,羽毛粉是按发酵液体积的7%(质量体积比,w/v)补加,进行高密度发酵3.5天,获得枯草芽孢杆菌HC2与发酵乳杆菌联合降解羽毛的羽毛降解液。该降解液中碱性角蛋白酶活力达到1087U/ml。
对照1:将发酵乳杆菌接种至MRS培养基,在36℃培养18h作为种子液,以体积比6%接种量转接至发酵培养基(500ml),在37℃和180r/min条件下通过分批补加葡萄糖和羽毛粉,其中葡萄糖是按发酵液体积的0.5%(质量体积比,w/v)补加,羽毛粉是按发酵液体积的7%(质量体积比,w/v)补加,进行高密度发酵3.5天,获得发酵乳杆菌的羽毛降解液。该降解液中碱性角蛋白酶活力达到324.4U/ml。
对照2:将枯草芽孢杆菌HC2挑至液体LB培养基在37℃和180r/min培养18h作为种子液,以体积比6%接种量转接至发酵培养基(500ml),在37℃和180r/min条件下通过分批补加葡萄糖和羽毛粉,其中葡萄糖是按发酵液体积的0.5%(质量体积比,w/v)补加,羽毛粉是按发酵液体积的7%(质量体积比,w/v)补加,进行高密度发酵3.5天,获得枯草芽孢杆菌HC2的羽毛降解液。该降解液中碱性角蛋白酶活力达到591.4U/ml。
由此可见,本发明的发酵乳杆菌和枯草芽孢杆菌HC2联合发酵获得的降解液中碱性角蛋白酶活力明显高于按照相同方法只接种枯草芽孢杆菌HC2或发酵乳杆菌降解羽毛粉的降解液中碱性角蛋白酶活力(见图1),说明这两种菌株的联合作用强于单菌作用。这也高于多数报道中优化前的碱性角蛋白酶活力(<260U/ml)。
向发酵乳杆菌与枯草芽孢杆菌HC2联合作用的羽毛降解液中加入降解液质量10%的甘蔗渣、降解液质量12%的麦麸和降解液质量10%的淀粉,混匀,在240r/min条件下造粒并制备高蛋白益生动物饲料,如图2所示。
饲料及制备过程相关指标的检测是采用凯氏定氮法(GB/T 6432-2018)测定饲料中的粗蛋白和可溶性蛋白,过滤法(GB/T 6434-2006)测定粗纤维含量,分光光度法检测角蛋白酶活力(全饲标[2021]52号)。通过梯度稀释培养法检测发酵液和饲料中的活性益生菌数量。
获得的饲料中粗蛋白含量为37.2%,粗纤维含量为6.3%,可溶性蛋白含量27.1g/l。两菌株联合作用的羽毛降解液中益生菌活菌数量为5.12×108cfu/ml,饲料中的活性益生菌数量为2.5×108cfu/g。
LB培养基:按质量分数计,酵母粉0.5%、胰蛋白胨1%、氯化钠1%,(固体LB培养基时需添加1.5%琼脂)pH 7.0,余量为水,其配制方法是将各成分混合均匀,121℃灭菌20min。
MRS培养基:按质量分数计,1%蛋白胨、0.5%牛肉浸粉、0.4%酵母浸粉、2%葡萄糖、0.2%柠檬酸三铵、0.2%K2HPO4、0.5%醋酸钠、0.04%MgSO4、0.005%MnSO4、0.1%吐温-80,余量为水,pH 6.2,其配制方法是将各成分混合均匀,121℃灭菌20min。
发酵培养基:按质量分数计,0.06%NaCl、0.05%KH2PO4、0.02%K2HPO4、0.06%MgSO4,6%膨化的羽毛,余量为水,pH为9.0,其配制方法是将各成分混合均匀,121℃灭菌20min。
Claims (8)
1.一种利用废弃羽毛制备高蛋白益生动物饲料的方法,其特征在于,是膨化的羽毛先经乳酸菌发酵,乳酸菌发酵液灭活后接种枯草芽孢杆菌种子液,通过分批补加葡萄糖和膨化的羽毛进行生物发酵和降解,获得降解羽毛的混合物;
在混合物中添加甘蔗渣、麦麸、淀粉,复配混匀后造粒,制得高蛋白益生动物饲料。
2.根据权利要求1所述的方法,其特征在于,所述的利用废弃羽毛制备高蛋白益生动物饲料的方法,具体为:
1)将乳酸菌接种于MRS培养基,在36℃培养16~20h,以体积比2%的接种量接种至发酵培养基,在36℃培养2~3天,结束后在60℃热处理2h;
所述的发酵培养基:按质量分数计,包括0.01~0.09%NaCl、0.02~0.1%KH2PO4、0.01~0.09%K2HPO4、0.01~0.09%MgSO4,6~8%膨化的羽毛,余量为水,pH为8.5~9.5,121℃灭菌20min;
2)将枯草芽孢杆菌接种于液体LB培养基,在37℃和160~240r/min条件下培养10~18h,制得种子液;
3)将种子液以体积比2~10%的接种量接种至步骤1)热处理后的发酵培养基中,在37℃和160~240r/min条件下通过分批补加葡萄糖和膨化的羽毛,其中葡萄糖是按发酵液体积的0.5%(质量体积比,w/v)补加,膨化的羽毛是按发酵液体积的5~8%(质量体积比,w/v)补加,进行高密度发酵和降解羽毛2~5天,获得降解羽毛的混合物;
4)将步骤3)混合物与混合物质量的8~12%甘蔗渣、混合物质量的9~15%麦麸、混合物质量的8~12%淀粉复配混匀,在220~240r/min条件下造粒,制得高蛋白益生动物饲料。
3.根据权利要求1或2所述的方法,其特征在于,所述的乳酸菌包括发酵乳杆菌、副干酪乳杆菌或植物乳杆菌等。
4.根据权利要求1或2所述的方法,其特征在于,所述的枯草芽孢杆菌是枯草芽孢杆菌(Bacillus subtilis)HC2。
5.根据权利要求1或2所述的方法,其特征在于,所述的废弃羽毛为鸡毛、鸭毛或鹅毛。
6.根据权利要求1或2所述的方法,其特征在于,所述的发酵培养基:按质量分数计,0.06%NaCl、0.05%KH2PO4、0.02%K2HPO4、0.06%MgSO4,6%膨化的羽毛,pH为9.0,121℃灭菌20min。
7.根据权利要求1或2所述的方法,其特征在于,芽孢杆菌与乳酸菌的联合作用降解废弃羽毛,制备高蛋白益生动物饲料。
8.根据权利要求1或2所述的方法,其特征在于,制得饲料的粗蛋白含量达到37.2%,粗纤维含量在6.3%左右,可溶性蛋白含量达到27.1g/l,益生菌活菌数达到2.5×108cfu/g。
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