CN114921513B - 一种乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法 - Google Patents
一种乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法 Download PDFInfo
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Abstract
本发明涉及一种乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法,火麻粕通过粉碎、微生物接种、加酶混合、固态发酵、多肽提取制备、冷冻干燥等步骤获得火麻蛋白肽粉。本发明利用乳酸菌与嗜冷菌来源的蛋白酶协同进行固态发酵制备火麻蛋白多肽,一方面乳酸菌在固态发酵过程中能够产生蛋白酶、氨肽酶等多种酶类,有效水解火麻蛋白,且微生物的发酵作用能够降低单独酶解的苦味,并且能够产生胞外多糖等活性代谢产物;另一方面来源于嗜冷菌的蛋白酶的加入能够辅助进行蛋白降解,显著加快火麻蛋白的降解,提高火麻蛋白的降解效率。本发明提供的乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法,工艺简单,制备效率高,适宜工业化生产。
Description
技术领域
本发明属于微生物酶解发酵技术领域,尤其是涉及一种乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法。
背景技术
火麻仁别名***仁、麻仁,在国内有一千多年的食用历史,《本草纲目》中记载麻仁,甘、平、无毒。现代研究表明,火麻仁有较好的利尿、抗炎、降血压以及降血脂等生能力活性。火麻仁富含蛋白、油脂、膳食纤维、微量元素以及氨基酸等营养成分。火麻仁中蛋白质含量为25%,含有20种氨基酸,包括9种人体必需氨基酸。火麻仁蛋白中80%为麻仁球蛋白,20%约为白蛋白,是一种优良的植物蛋白,富含有精氨酸、谷氨酸、组氨酸等功能性氨基酸。火麻蛋白工业上主要从火麻籽榨油后的副产物火麻粕中获取,但是火麻蛋白结构紧密,尤其是加热后火麻蛋白因为变性导致溶解度下降,降低消化率,生物效价并不理想,需要寻找一种提高火麻蛋白生物利用效率的方法。
目前火麻蛋白主要通过酸碱水解或是酶解法获得火麻蛋白肽,酸碱水解条件较为剧烈,容易破坏水解多肽的构型以及活性,并且会产生有害副产物;酶水解法是目前火麻蛋白肽的主要制备方法,采用中性蛋白酶、木瓜蛋白酶等进行酶解,但是单纯的酶对火麻蛋白水解程度不高,酶解后的水解物中存在较多苦味肽,导致风味与口感不好。此外单纯酶解的成本较高,大规模工业化受到限制。
发明内容
本发明的目的就是为了提供一种乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法,以解决单纯的微生物固态发酵获得火麻蛋白肽效率较低的问题.
本发明提供的方法工艺简单,制备条件温和,适宜于大规模工业化生产。
本发明的目的可以通过以下技术方案来实现:
本发明提供一种乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法,利用具有产蛋白酶能力的乳酸菌与嗜冷菌蛋白酶协同进行火麻粕固态发酵,制备火麻蛋白肽。
在本发明的一个实施方式中,所述方法包括以下步骤:
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;
(2)将乳酸菌活化,制备乳酸菌发酵剂;
(3)将制备的乳酸菌发酵剂与嗜冷菌蛋白酶接入火麻粕粉中进行固态发酵;
(4)固态发酵结束后,将发酵火麻粕粉进行灭酶处理;
(5)将灭酶后的火麻粕粉进行热水浸提,离心去除沉淀,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉。
进一步的,在本发明的一个实施方式中,步骤(1)中,火麻粕粉的粒径为300-900μm。
进一步的,在本发明的一个实施方式中,步骤(1)中,火麻粕粉碎后经过121℃高温蒸汽灭菌处理。
进一步的,在本发明的一个实施方式中,步骤(2)中,乳酸菌发酵剂制备方法为:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O0.25g/L;培养基配置好了后进行高温灭菌;
b:将乳酸菌活化后接种至培养基中,接种量为3%,培养20h后离心收集菌体,然后加入无菌水调节菌浓为1×109/mL,即为乳酸菌发酵剂。
进一步的,在本发明的一个实施方式中,步骤a中,进行高温灭菌的温度为121℃。
进一步的,在本发明的一个实施方式中,步骤(2)中,乳酸菌选择为唾液乳杆菌、植物乳杆菌、发酵乳杆菌或短乳杆菌中的一种或者几种。
进一步的,在本发明的一个实施方式中,步骤(3)中,加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在60%-85%(w/w),无菌营养液中含有2%-15%的葡萄糖。
进一步的,在本发明的一个实施方式中,步骤(3)中,乳酸菌发酵剂接种量为5%-30%(v/w);嗜冷菌蛋白酶添加量为30-220U/g,乳酸菌与嗜冷菌蛋白酶可以同步加入火麻粕粉中,也可以分步加入。
进一步的,在本发明的一个实施方式中,步骤(3)中,所述嗜冷菌蛋白酶为一种金属蛋白酶,其分离于荧光假单胞菌BNCC232070,为市售的成熟产品,菌株购买自商城北纳创联生物科技有限公司。其氨基酸序列如SEQ ID NO.1所示,具体如下:
MNGIPPSPTPLRLSAARVSQYEPEEETDAGKSVKMSLDTFQAGRIISRNGAVWGDQNRDGHTTIYYKFGQHNYTDQHDINTNFTESQKNGLRLAMQSWSDIANVAYKEKPPKGEWVDDGKSGKDTRDAEATLSLLKRVNSHDPGGYAGIGTSHVGITFRYAGESTLPNTEFAGVAIHELGHHLGLPHPGNYNGRGSDYEKDAAYNEDTIAHSIMSYFSEKNNGYDFKGSVVRTPMMDDISASQRIYGANYNTRNTDTTYGFNSNTGREALTFKSSRDRPVLCIWDGGGNDTLDCSKFAADQQINLNEQTFSNVAGLKGNISIARGVTLENAVGGHGNDQILGNDADNRLTGGVGADQLTGGKGKDTFIYKDISDSTPEKPDLITDFVSGTDSFDLSAMLKKNGLKDLHFVSQFSGRAGEALLDFDEDRNLGRLKVDLSGKGRENFMVTTNGRIYPSDVLSTPSLHKTVDTSPPRFTPDGARTDGYVPPPPPPPLVLPPTVKFMTGTQGNDVLRGDHGDNNITGGAGADVLFGGAGKDIFIYEKASDSSVKRPDQILDFTTGIDKIDVSKALQAAGTDTLIFVEKFTGRIGETVIRYNAKNKESSLAVDMTGNGKADLHIVSRGQIKADDIISK。
进一步的,在本发明的一个实施方式中,步骤(3)中,固态发酵温度为28-45℃,发酵时间为1-4天。
进一步的,在本发明的一个实施方式中,步骤(4)中,进行灭酶处理的条件为:100℃水浴灭酶20min。
进一步的,在本发明的一个实施方式中,步骤(5)中,将去离子水加入发酵火麻粕中进行搅拌提取,固液比为1:5-1:25(w/v),提取温度为20-70℃,提取时间为40-240min,提取2次,合并提取液。
进一步的,在本发明的一个实施方式中,步骤(6)中,采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,采用超滤膜超滤制备不同分子量活性肽,超滤膜的截留分子量分别为10k道尔顿。
与现有技术相比,本发明提供的制备火麻蛋白肽的方法,能够直接以火麻粕为原料进行固态发酵,获得不同分子量的的火麻蛋白肽,生产工艺简单、原料成本低、生产效率高,容易实现大规模工业化生产。本发明采用乳酸菌作为发酵剂进行固态发酵,一方面乳酸菌作为兼性厌氧菌,适用于大批量固体物料的静置培养,另一方面乳酸菌能够合成蛋白酶、氨肽酶等多种酶类水解火麻蛋白,提高火麻蛋白的生物利用度。与酶解法相比,本发明提供的乳酸菌与嗜冷菌蛋白酶联合固态发酵方法,显著提高了活性肽制备效率,同时能够显著降低水解肽的苦味。
具体实施方式
下面结合具体实施例对本发明进行详细说明。本实施例以本发明技术方案为前提进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
以下实施例中的唾液乳杆菌、植物乳杆菌、发酵乳杆菌或短乳杆菌均为本领域常规生物材料,以下实施例中嗜冷菌蛋白酶具体为一种金属蛋白酶,其分离于荧光假单胞菌BNCC232070,为市售的成熟产品,菌株购买自商城北纳创联生物科技有限公司。其氨基酸序列如SEQ ID NO.1所示。
实施例1:乳酸菌固态发酵火麻粕生产火麻蛋白肽(对照实验)
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;火麻粕粉的粒径为500μm,火麻粕粉碎后经过121℃高温蒸汽灭菌;
(2)将唾液乳杆菌活化,制备唾液乳杆菌发酵剂;
制备唾液乳杆菌发酵剂的步骤如下:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O0.25g/L;培养基配置好了后进行121℃高温灭菌;
b:将唾液乳杆菌活化后接种至培养基中,接种量为3%,培养20h后离心收集菌体,然后加入无菌水调节菌浓为1×109/mL,即为唾液乳杆菌发酵剂;
(3)加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在75%(w/w),无菌营养液中含有10%的葡萄糖;将制备的唾液乳杆菌发酵剂接入火麻粕粉中进行固态发酵;唾液乳杆菌发酵剂接种量为15%(v/w);固态发酵温度为37℃,发酵时间为3天;
(4)固态发酵结束后,将发酵火麻粕粉进行100℃水浴灭酶20min;
(5)将去离子水加入灭酶后的火麻粕粉进行热水搅拌浸提,固液比为1:15(w/v),提取温度为50℃,提取时间为150min,离心去除沉淀,提取2次,合并上清提取液,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉;采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,然后12000g高速离心去除杂质,采用截留分子量为10k道尔顿的超滤膜超滤制备多肽,截留后的多肽溶液经过冷冻干燥后得到具有抗氧化性的火麻蛋白肽粉,肽粉的蛋白质含量为80%(w/w),分子量为1k道尔顿-5k道尔顿的多肽占57%,多肽的DPPH清除率达到66.3%。
实施例2:乳酸菌与嗜冷菌蛋白酶同步固态发酵火麻粕生产火麻蛋白肽
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;火麻粕粉的粒径为300μm,火麻粕粉碎后经过121℃高温蒸汽灭菌;
(2)将唾液乳杆菌活化,制备唾液乳杆菌发酵剂;
制备唾液乳杆菌发酵剂的步骤如下:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O0.25g/L;培养基配置好了后进行121℃高温灭菌;
b:将唾液乳杆菌活化后接种至培养基中,接种量为3%,培养20h后离心收集菌体,然后加入无菌水调节菌浓为1×109/mL,即为唾液乳杆菌发酵剂;
(3)将制备的唾液乳杆菌发酵剂与嗜冷菌蛋白酶同步接入火麻粕粉中进行固态发酵;加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在60%(w/w),无菌营养液中含有2%的葡萄糖;唾液乳杆菌发酵剂接种量为5%(v/w),嗜冷菌蛋白酶添加量为30U/g,固态发酵温度为28℃,发酵时间为1天;
(4)固态发酵结束后,将发酵火麻粕粉进行100℃水浴灭酶20min;
(5)将去离子水加入灭酶后的火麻粕粉进行热水搅拌浸提,固液比为1:5(w/v),提取温度为20℃,提取时间为40min,离心去除沉淀,提取2次,合并上清提取液,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉;采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,然后12000g高速离心去除杂质,采用截留分子量为10k道尔顿的超滤膜超滤制备多肽,截留后的多肽溶液经过冷冻干燥后得到具有抗氧化性的火麻蛋白肽粉,肽粉的蛋白质含量为83%(w/w),分子量为1k道尔顿-5k道尔顿的多肽占61%,多肽的DPPH清除率达到78.5%。
实施例3:乳酸菌与嗜冷菌蛋白酶同步固态发酵火麻粕生产火麻蛋白肽
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;火麻粕粉的粒径为500μm,火麻粕粉碎后经过121℃高温蒸汽灭菌;
(2)将唾液乳杆菌活化,制备唾液乳杆菌发酵剂;
制备唾液乳杆菌发酵剂的步骤如下:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O0.25g/L;培养基配置好了后进行121℃高温灭菌;
b:将唾液乳杆菌活化后接种至培养基中,接种量为3%,培养20h后离心收集菌体,然后加入无菌水调节菌浓为1×109/mL,即为唾液乳杆菌发酵剂;
(3)将制备的唾液乳杆菌发酵剂与嗜冷菌蛋白酶同步接入火麻粕粉中进行固态发酵;加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在75%(w/w),无菌营养液中含有10%的葡萄糖;唾液乳杆菌发酵剂接种量为15%(v/w),嗜冷菌蛋白酶添加量为150U/g,固态发酵温度为37℃,发酵时间为3天;
(4)固态发酵结束后,将发酵火麻粕粉进行100℃水浴灭酶20min;
(5)将去离子水加入灭酶后的火麻粕粉进行热水搅拌浸提,固液比为1:15(w/v),提取温度为50℃,提取时间为150min,离心去除沉淀,提取2次,合并上清提取液,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉;采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,然后12000g高速离心去除杂质,采用截留分子量为10k道尔顿的超滤膜超滤制备多肽,截留后的多肽溶液经过冷冻干燥后得到具有抗氧化性的火麻蛋白肽粉,肽粉的蛋白质含量为90%(w/w),分子量为1k道尔顿-5k道尔顿的多肽占71%,多肽的DPPH清除率达到94.9%。
实施例4:乳酸菌与嗜冷菌蛋白酶同步固态发酵火麻粕生产火麻蛋白肽
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;火麻粕粉的粒径为500μm,火麻粕粉碎后经过121℃高温蒸汽灭菌;
(2)将植物乳杆菌活化,制备植物乳杆菌发酵剂;
制备植物乳杆菌发酵剂的步骤如下:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O0.25g/L;培养基配置好了后进行121℃高温灭菌;
b:将植物乳杆菌活化后接种至培养基中,接种量为3%,培养20h后离心收集菌体,然后加入无菌水调节菌浓为1×109/mL,即为植物乳杆菌发酵剂;
(3)将制备的植物乳杆菌发酵剂与嗜冷菌蛋白酶同步接入火麻粕粉中进行固态发酵;加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在75%(w/w),无菌营养液中含有10%的葡萄糖;植物乳杆菌发酵剂接种量为15%(v/w),嗜冷菌蛋白酶添加量为150U/g,固态发酵温度为37℃,发酵时间为3天;
(4)固态发酵结束后,将发酵火麻粕粉进行100℃水浴灭酶20min;
(5)将去离子水加入灭酶后的火麻粕粉进行热水搅拌浸提,固液比为1:15(w/v),提取温度为50℃,提取时间为150min,离心去除沉淀,提取2次,合并上清提取液,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉;采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,然后12000g高速离心去除杂质,采用截留分子量为10k道尔顿的超滤膜超滤制备多肽,截留后的多肽溶液经过冷冻干燥后得到具有抗氧化性的火麻蛋白肽粉,肽粉的蛋白质含量为85%(w/w),分子量为1k道尔顿-5k道尔顿的多肽占65%,多肽的DPPH清除率达到91.2%。
实施例5:乳酸菌与嗜冷菌蛋白酶同步固态发酵火麻粕生产火麻蛋白肽
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;火麻粕粉的粒径为900μm,火麻粕粉碎后经过121℃高温蒸汽灭菌;
(2)将唾液乳杆菌活化,制备唾液乳杆菌发酵剂;
制备唾液乳杆菌发酵剂的步骤如下:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O0.25g/L;培养基配置好了后进行121℃高温灭菌;
b:将唾液乳杆菌活化后接种至培养基中,接种量为3%,培养20h后离心收集菌体,然后加入无菌水调节菌浓为1×109/mL,即为唾液乳杆菌发酵剂;
(3)将制备的唾液乳杆菌发酵剂与嗜冷菌蛋白酶同步接入火麻粕粉中进行固态发酵;加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在85%(w/w),无菌营养液中含有15%的葡萄糖;唾液乳杆菌发酵剂接种量为30%(v/w),嗜冷菌蛋白酶添加量为220U/g,固态发酵温度为45℃,发酵时间为4天;
(4)固态发酵结束后,将发酵火麻粕粉进行100℃水浴灭酶20min;
(5)将去离子水加入灭酶后的火麻粕粉进行热水搅拌浸提,固液比为1:25(w/v),提取温度为70℃,提取时间为240min,离心去除沉淀,提取2次,合并上清提取液,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉;采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,然后12000g高速离心去除杂质,采用截留分子量为10k道尔顿的超滤膜超滤制备多肽,截留后的多肽溶液经过冷冻干燥后得到具有抗氧化性的火麻蛋白肽粉,肽粉的蛋白质含量为85%(w/w),分子量为1k道尔顿-5k道尔顿的多肽占62%,多肽的DPPH清除率达到82.2%。
实施例6:乳酸菌与嗜冷菌蛋白酶分步固态发酵火麻粕生产火麻蛋白肽
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;火麻粕粉的粒径为500μm,火麻粕粉碎后经过121℃高温蒸汽灭菌;
(2)将唾液乳杆菌活化,制备唾液乳杆菌发酵剂;
制备唾液乳杆菌发酵剂的步骤如下:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O0.25g/L;培养基配置好了后进行121℃高温灭菌;
b:将唾液乳杆菌活化后接种至培养基中,接种量为3%,培养20h后离心收集菌体,然后加入无菌水调节菌浓为1×109/mL,即为唾液乳杆菌发酵剂;
(3)将制备的唾液乳杆菌发酵剂与嗜冷菌蛋白酶分步接入火麻粕粉中进行固态发酵;加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在85%(w/w),无菌营养液中含有15%的葡萄糖;唾液乳杆菌发酵剂接种量为15%(v/w),发酵温度为37℃,发酵1天后,按150U/g加入嗜冷菌蛋白酶,搅拌均匀后相同条件下继续发酵2天;
(4)固态发酵结束后,将发酵火麻粕粉进行100℃水浴灭酶20min;
(5)将去离子水加入灭酶后的火麻粕粉进行热水搅拌浸提,固液比为1:25(w/v),提取温度为70℃,提取时间为240min,离心去除沉淀,提取2次,合并上清提取液,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉;采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,然后12000g高速离心去除杂质,采用截留分子量为10k道尔顿的超滤膜超滤制备多肽,截留后的多肽溶液经过冷冻干燥后得到具有抗氧化性的火麻蛋白肽粉,肽粉的蛋白质含量为86%(w/w),分子量为1k道尔顿-5k道尔顿的多肽占67%,多肽的DPPH清除率达到89.8%。
实施例7:乳酸菌与嗜冷菌蛋白酶分步固态发酵火麻粕生产火麻蛋白肽
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;火麻粕粉的粒径为500μm,火麻粕粉碎后经过121℃高温蒸汽灭菌;
(2)将植物乳杆菌活化,制备植物乳杆菌发酵剂;
制备植物乳杆菌发酵剂的步骤如下:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O0.25g/L;培养基配置好了后进行121℃高温灭菌;
b:将植物乳杆菌活化后接种至培养基中,接种量为3%,培养20h后离心收集菌体,然后加入无菌水调节菌浓为1×109/mL,即为植物乳杆菌发酵剂;
(3)将制备的植物乳杆菌发酵剂与嗜冷菌蛋白酶分步接入火麻粕粉中进行固态发酵;加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在85%(w/w),无菌营养液中含有15%的葡萄糖;植物乳杆菌发酵剂接种量为15%(v/w),发酵温度为37℃,发酵1天后,按150U/g加入嗜冷菌蛋白酶,搅拌均匀后相同条件下继续发酵2天;
(4)固态发酵结束后,将发酵火麻粕粉进行100℃水浴灭酶20min;
(5)将去离子水加入灭酶后的火麻粕粉进行热水搅拌浸提,固液比为1:25(w/v),提取温度为70℃,提取时间为240min,离心去除沉淀,提取2次,合并上清提取液,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉;采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,然后12000g高速离心去除杂质,采用截留分子量为10k道尔顿的超滤膜超滤制备多肽,截留后的多肽溶液经过冷冻干燥后得到具有抗氧化性的火麻蛋白肽粉,肽粉的蛋白质含量为82%(w/w),分子量为1k道尔顿-5k道尔顿的多肽占63%,多肽的DPPH清除率达到87.5%。
下表是本发明的实施例与对照例的实验效果对比
综上所述,本发明采用乳酸菌作为发酵剂进行固态发酵,一方面乳酸菌作为兼性厌氧菌,适用于大批量固体物料的静置培养,另一方面乳酸菌能够合成蛋白酶、氨肽酶等多种酶类水解火麻蛋白,提高火麻蛋白的生物利用度。与酶解法相比,本专利提供的乳酸菌与嗜冷菌蛋白酶联合固态发酵方法,显著提高了活性肽制备效率,同时能够显著降低水解肽的苦味。本发明提供的制备火麻蛋白肽的方法,能够直接以火麻粕为原料进行固态发酵,获得不同分子量的火麻蛋白肽,生产工艺简单、原料成本低、生产效率高,容易实现大规模工业化生产。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 上海理工大学
<120> 一种乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 633
<212> PRT
<213> 荧光假单胞菌(Pseudomonas fluorescens )
<400> 1
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Thr Thr Tyr Gly Phe Asn Ser Asn Thr Gly Arg Glu Ala Leu Thr Phe
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Claims (1)
1.一种乳酸菌与嗜冷菌蛋白酶组合固态发酵制备火麻蛋白肽的方法,其特征在于,利用具有产蛋白酶能力的乳酸菌与嗜冷菌蛋白酶协同进行火麻粕固态发酵,制备火麻蛋白肽,所述方法包括以下步骤:
(1)将压榨制油之后的火麻粕粉碎,得到火麻粕粉;
(2)将乳酸菌活化,制备乳酸菌发酵剂;
(3)将制备的乳酸菌发酵剂与嗜冷菌蛋白酶接入火麻粕粉中进行固态发酵;
(4)固态发酵结束后,将发酵火麻粕粉进行灭酶处理;
(5)将灭酶后的火麻粕粉进行热水浸提,离心去除沉淀,收集上清获得火麻蛋白肽粗提液;
(6)将火麻蛋白肽粗提液进行脱色、脱苦、分离纯化以及冷冻干燥获得火麻蛋白肽粉;
步骤(2)中,乳酸菌发酵剂制备方法为:
a:配置培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4•7H2O 0.58g/L,MnSO4•4H2O 0.25g/L;培养基配置好了后进行高温灭菌;
b:将乳酸菌活化后接种至培养基中,接种量为3%,培养后离心收集菌体,然后加入无菌水调节菌浓,即为乳酸菌发酵剂;
步骤(2)中,乳酸菌选择为唾液乳杆菌或植物乳杆菌;
步骤(3)中,所述嗜冷菌蛋白酶的氨基酸序列如SEQ ID NO.1所示;
步骤(1)中,火麻粕粉的粒径为300-900 μm,步骤(1)中,火麻粕粉碎后经过高温蒸汽灭菌处理;
步骤(3)中,加入无菌营养液至火麻粕粉中,控制火麻粕粉含水量在75%-85%(w/w),无菌营养液中含有10%-15%的葡萄糖;步骤(3)中,乳酸菌发酵剂接种量为15%(v/w);嗜冷菌蛋白酶添加量为150 U/g;乳酸菌与嗜冷菌蛋白酶同步加入火麻粕粉中,或分步加入;步骤(3)中,固态发酵温度为37-45℃,发酵时间为3-4天;
步骤(5)中,将去离子水加入发酵火麻粕中进行搅拌提取,固液比为1:15-1:25(w/v),提取温度为50-70℃,提取时间为150-240 min,提取2次,合并提取液;
步骤(6)中,采用活性炭对火麻蛋白肽粗提液进行脱色、脱苦,采用超滤膜超滤制备不同分子量活性肽,超滤膜的截留分子量为10 k道尔顿。
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