CN111406899A - 一种具有高抗氧化活性的发酵血肠制作方法 - Google Patents

一种具有高抗氧化活性的发酵血肠制作方法 Download PDF

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CN111406899A
CN111406899A CN202010276904.2A CN202010276904A CN111406899A CN 111406899 A CN111406899 A CN 111406899A CN 202010276904 A CN202010276904 A CN 202010276904A CN 111406899 A CN111406899 A CN 111406899A
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blood
livestock
poultry
meat
sausage
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李思宁
唐善虎
夏佳军
蔡寅川
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Industrial Economy Research Institute Of ABA Tibetan And Qiang Autonomous Prefecture Service Center For Small And Medium Sized Enterprises Of ABA Tibetan And Qiang Autonomous Prefecture
Southwest Minzu University
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Industrial Economy Research Institute Of ABA Tibetan And Qiang Autonomous Prefecture Service Center For Small And Medium Sized Enterprises Of ABA Tibetan And Qiang Autonomous Prefecture
Southwest Minzu University
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Priority to CN202010276904.2A priority Critical patent/CN111406899A/zh
Publication of CN111406899A publication Critical patent/CN111406899A/zh
Priority to CN202110280386.6A priority patent/CN112914041B/zh
Priority to US17/221,804 priority patent/US11730180B2/en
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Abstract

本发明的目的在于提供一种具有高抗氧化活性的发酵血肠及其制作方法，包括以下步骤：(1)Plastein反应制备血红蛋白抗氧化肽，(2)畜禽肉腌制和一次发酵处理，(3)畜禽血脱腥处理及破碎，(4)灌制及二次发酵，(5)干燥后获得成品。与现有技术相比，本发明使用了血液Plastein反应制备的高活性抗氧化肽，产品发酵血肠的总抗氧化能力达到6.89U/mg，增加率为58％；对血液进行了脱腥，且乳酸菌与酵母菌联合二次发酵游离氨基酸总量达到12.74mg/g，增加率为42％，使制作得到的血肠风味更好，咀嚼性更高，同时也为畜禽血液资源的综合利用提供了新途径。

Description

一种具有高抗氧化活性的发酵血肠制作方法
技术领域
本发明涉及一种肉制品及其加工方法，尤其涉及一种具有高抗氧化活性的发酵血肠及其制作方法。
背景技术
血液作为畜禽屠宰加工中的主要副产物，是十分理想的蛋白质资源。我国每年屠宰畜禽产生的血液，仅25％进行了集中合理利用，绝大多数被当作废弃物排放掉，这不仅浪费了资源，也污染了环境。
Plastein反应中，在酶促胁迫下，添加理想的外源性氨基酸使其合成到新蛋白质中，可产生原料蛋白质所没有的新肽段。高丹丹等人用中性蛋白酶酶解泥鳅蛋白，在此基础之上用木瓜蛋白酶对得到的酶解产物进行Plastein反应修饰，并添加0.5mmol/g的His(组氨酸)，Plastein反应修饰产物对DPPH自由基的清除率高达77.98％。车丽辉添加Pro(脯氨酸)和Arg(精氨酸)两种外源氨基酸对沙蜇酶解肽进行Plastein反应改性，Plastein反应修饰物的DPPH的清除率和羟基自由基清除率都有提高。目前未见Plastein反应用于改善血液抗氧化肽的报道。
血肠是一种以畜禽肉和血液为主要原料，辅以其他原辅料制作而成的发酵或不发酵灌肠类肉制品。李福存研究了发酵鸭血肠生产工艺，该研究认为鸭血添加量25％，菌种接种量107cfu/g，在30℃发酵18h后的血肠品质最佳。贡佳欣等人确定发酵牦牛血肠的最优工艺参数条件为，牦牛血添加量20％，发酵温度25℃，发酵时间16h，发酵菌株接种量1.5％。中国专利CN 108835533 A公开了一种保质期长的血肠制备方法，该产品通过两段式发酵工艺而实现。这些研究都是将血液直接与畜禽肉混合制作血肠，没有涉及到血液的脱腥及其中生物活性物质的有效利用，且也未涉及到益生酵母菌的发酵。
发明内容
本发明的目的在于提供一种脱腥，耐保存，风味更好，具有高抗氧化活性畜禽肉发酵血肠的制作方法。
本发明的技术方案是：
一种具有高抗氧化活性的发酵血肠制作方法，其特征在于，包括以下步骤：
(1)Plastein反应制备血红蛋白抗氧化肽：
收集新鲜畜禽血，将占新鲜畜禽血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，将抗凝血再4000r/min离心15min，收集沉淀的血细胞，取沉淀的血细胞溶于体积比为0.6-0.8倍的蒸馏水中，经40kHz超声破碎10min后，调节pH值至2.5作为血细胞反应底物；按重量比，血细胞反应底物∶胃蛋白酶＝3000∶1添加胃蛋白酶，并在37℃下酶解4.0-7.5h；经4000r/min离心15min后，取上清为血细胞肽液；按血细胞肽液重量的1％加入L-脯氨酸(Pro)和1％加入L-组氨酸(His)，调节pH值至7.5，30℃保温4.0-6.0h后，冷却至室温，真空浓缩至固形物含量为50％左右，获得血红蛋白抗氧化肽备用；胃蛋白酶活力为10万U/g；
(2)畜禽肉腌制和一次发酵处理：
将去掉脂肪、筋膜、肌腱的新鲜畜禽瘦肉，切成5cm见方的肉块，按照重量比，每100份肉加入葡萄糖2.00-3.00份、食盐3.00-4.00份、五香粉0.12-0.25份、味精0.05-0.08份、焦磷酸钠0.20-0.40份，搅拌均匀，放于4℃腌制8-12h；
将活化好的肉葡萄球菌(Staphylococcus carnosus)菌液按每克新鲜畜禽瘦肉放入106cfu与腌制肉块混匀，密封后在32-37℃发酵24h；获得一次发酵畜禽瘦肉备用；
(3)畜禽血脱腥处理及破碎：
收集新鲜畜禽血，将占新鲜畜禽血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，调节pH至5.0-5.5，将抗凝血体积比0.1％的酸性脂肪酶加入抗凝血，于37℃酶解30min，再95℃灭酶活性5min，获得脱腥血液；因热处理后脱腥血液已凝固，需将脱腥血液破碎成2.5-3.0cm见方的脱腥血液小颗粒备用；酸性脂肪酶活力为10万U/g；
(4)灌制及二次发酵：
将重量比血红蛋白抗氧化肽∶一次发酵畜禽瘦肉∶脱腥血液小颗粒＝0.2∶1.0∶4.0混合成为肽肉血混物，按每克肽肉血混物放入107cfu***克鲁维酵母(Kluyveromycesmarxianus)和鼠李糖乳杆菌(Lactobacillus rhamnosus)的混合菌液，并搅拌均匀，最后灌入天然的猪小肠肠衣，用温水洗净香肠表面的油污，将其自然悬挂在恒温培养箱内，于35℃恒温发酵18h；在107cfu混合菌液中，***克鲁维酵母∶鼠李糖乳杆菌＝1.0∶2.5；
(5)干燥后获得成品：
发酵后的香肠置于50℃干燥箱，干燥至含水量≤25％，即为成品发酵血肠。
畜禽血为猪血、牛血、牦牛血、鸡血、鸭血中的一种或几种。
本发明所述的酸性脂肪酶是由上海源叶生物科技有限公司生产，活力为10万U/g。
本发明的优点：
本发明将血细胞用胃蛋白酶处理，生成了更多的小分子肽，且获得的水解产物具有更好的凝胶性、热稳定性和乳化性。再通过Plastein反应修饰的方法有效提高了肽的抗氧化活性，制得产物的DPPH(1，1-二苯基-2-三硝基苯肼)自由基清除率从对照组的78％提高到90％以上，总抗氧化能力增加58％，为畜禽血液资源的综合利用提供了新途径。
本发明利用酸性脂肪酶处理血液，有效去除了血液中的部分胺类、醛类和醇类物质，对血液起到了较好的脱腥效果。
本发明使用肉葡萄球菌先发酵畜禽肉，然后采用鼠李糖乳杆菌和***克鲁维酵母对畜禽肉、血液等进行再发酵处理，在降低产品pH延长保质期的同时，还赋予产品更好的风味。
具体实施方式
实施例1：用牦牛肉和牦牛血进行具有抗氧化活性的发酵血肠制作方法，包括以下步骤：
(1)Plastein反应制备血红蛋白抗氧化肽：
收集新鲜牦牛血，将占新鲜牦牛血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，将抗凝血再4000r/min离心15min，收集沉淀的血细胞，取沉淀的血细胞溶于体积比为0.8倍的蒸馏水中，经40kHz超声破碎10min后，调节pH值至2.5作为血细胞反应底物；按重量比，血细胞反应底物∶胃蛋白酶＝3000∶1添加胃蛋白酶，并在37℃下酶解7.5h；经4000r/min离心15min后，取上清为血细胞肽液；按血细胞肽液重量的1％加入L-脯氨酸(Pro)和1％加入L-组氨酸(His)，调节pH值至7.5，30℃保温4.0-6.0h后，冷却至室温，真空浓缩至固形物含量为50％左右，获得牦牛血红蛋白抗氧化肽备用；胃蛋白酶活力为10万U/g；
(2)畜禽肉腌制和一次发酵处理：
将去掉脂肪、筋膜、肌腱的新鲜牦牛肉，切成5cm见方的肉块，按照重量比，每100份肉加入葡萄糖3.00份、食盐3.50份、五香粉0.25份、味精0.07份、焦磷酸钠0.40份，搅拌均匀，放于4℃腌制8-12h；
将活化好的肉葡萄球菌(Staphylococcus carnosus)菌液按每克新鲜畜牦牛肉放入106cfu与腌制肉块混匀，密封后在32-37℃发酵24h；获得一次发酵牦牛肉备用；
(3)畜禽血脱腥处理及破碎：
收集新鲜牦牛血，将占新鲜牦牛血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，调节pH至5.0-5.5，将抗凝血体积比0.1％的酸性脂肪酶加入抗凝血，于37℃酶解30min，再95℃灭酶活性5min，获得脱腥血液；因热处理后脱腥血液已凝固，需将脱腥血液破碎成2.5-3.0cm见方的脱腥血液小颗粒备用；酸性脂肪酶活力为10万U/g；表1是本步骤用酸性脂肪酶处理后的新鲜牦牛血去腥味物质的检测对照表，该表中数据表明，用酸性脂肪酶处理后的新鲜牦牛血，大量腥味物质被消除，去腥味效果好。
表1 牦牛血中疑似腥味物质GC-MS检出情况(％)
序号 疑似腥味物质 对照组 酸性脂肪酶处理组
1 三甲胺 0.13 未检出
2 叔丁胺 2.34 0.91
3 4-庚胺 0.49 未检出
4 2-甲基庚醛 1.11 1.01
5 反式-4，5-环氧-(E)-2-癸烯醛 2.08 0.09
6 3-甲基-1-戊醇 0.99 未检出
7 反-2-癸烯醇 0.22 未检出
8 2-甲基吡嗪 0.22 0.34
9 2，5-二甲基吡嗪 0.14 0.08
(4)灌制及二次发酵：
将重量比血红蛋白抗氧化肽∶一次发酵畜禽瘦肉∶脱腥血液小颗粒＝0.2∶1.0∶4.0混合成为肽肉血混物，按每克肽肉血混物放入107cfu***克鲁维酵母(Kluyveromycesmarxianus)和鼠李糖乳杆菌(Lactobacillus rhamnosus)的混合菌液，并搅拌均匀，最后灌入天然的猪小肠肠衣，用温水洗净香肠表面的油污，将其自然悬挂在恒温培养箱内，于35℃恒温发酵18h；在107cfu混合菌液中，***克鲁维酵母∶鼠李糖乳杆菌＝1.0∶2.5；
表2是本步骤处理后，形成风味的前体物质游离氨基酸总量12.74mg/g，比不发酵组增加了3.76mg/g，增长率为42％，使产品风味更浓郁，pH值更低，抑制了有害菌生长，可增加产品的保质期。
表2 鼠李糖乳杆菌和***克鲁维酵母发酵后血肠的理化指标
理化指标 对照组 乳酸菌组 乳酸菌+酵母菌发酵组
pH 5.21 4.75 4.69
游离氨基酸总量(mg/g) 8.98 10.71 12.74
硫代巴比妥酸(mg/100g) 0.246 0.251 0.248
咀嚼性(g) 879.40 1068.01 1176.19
感官评分 76.04 81.00 82.20
注：硫代巴比妥酸值是表征脂肪氧化程度的指标。
(5)干燥后获得成品：
发酵后的香肠置于50℃干燥箱，干燥至含水量≤25％，即为成品发酵血肠。表3是本实施例的制作方法所制成的发酵血肠其总抗氧化能力增加了2.53U/mg，增加率为58％，而DPPH自由基清除率增加了17.71％，增加率为23％，提高抗氧化效果十分显著。
表3 牦牛血液Plastein反应物加入后对牦牛发酵血肠的抗氧化能力影响
Figure BSA0000206058540000051
实施例2：用猪肉和猪血进行具有高抗氧化活性的发酵血肠制作方法，包括以下步骤：
(1)Plastein反应制备血红蛋白抗氧化肽：
收集新鲜猪血，将占新鲜猪血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，将抗凝血再4000r/min离心15min，收集沉淀的血细胞，取沉淀的血细胞溶于体积比为0.7倍的蒸馏水中，经40kHz超声破碎10min后，调节pH值至2.5作为血细胞反应底物；按重量比，血细胞反应底物∶胃蛋白酶＝3000∶1添加胃蛋白酶，并在37℃下酶解7.5h；经4000r/min离心15min后，取上清为血细胞肽液；按血细胞肽液重量的1％加入L-脯氨酸(Pro)和1％加入L-组氨酸(His)，调节pH值至7.5，30℃保温5.0h后，冷却至室温，真空浓缩至固形物含量为50％左右，获得猪血红蛋白抗氧化肽备用；胃蛋白酶活力为10万U/g；
(2)畜禽肉腌制和一次发酵处理：
将去掉脂肪、筋膜、肌腱的新鲜牦牛肉，切成5cm见方的肉块，按照重量比，每100份肉加入葡萄糖3.00份、食盐3.00份、五香粉0.20份、味精0.07份、焦磷酸钠0.30份，搅拌均匀，放于4℃腌制10h；
将活化好的肉葡萄球菌(Staphylococcus carnosus)菌液按每克新鲜畜牦牛肉放入106cfu与腌制肉块混匀，密封后在32-37℃发酵24h；获得一次发酵猪肉备用；
(3)畜禽血脱腥处理及破碎：
收集新鲜猪血，将占新鲜猪血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗
凝，获得抗凝血，调节pH至5.0-5.5，将抗凝血体积比0.1％的酸性脂肪酶加入抗凝血，于37℃酶解30min，再95℃灭酶活性5min，获得脱腥血液；因热处理后脱腥血液已凝固，需将脱腥血液破碎成2.5-3.0cm见方的脱腥血液小颗粒备用；酸性脂肪酶活力为10万U/g；
(4)灌制及二次发酵：
将重量比血红蛋白抗氧化肽∶一次发酵畜禽瘦肉∶脱腥血液小颗粒＝0.2∶1.0∶4.0混合成为肽肉血混物，按每克肽肉血混物放入107cfu***克鲁维酵母(Kluyveromycesmarxianus)和鼠李糖乳杆菌(Lactobacillus rhamnosus)的混合菌液，并搅拌均匀，最后灌入天然的猪小肠肠衣，用温水洗净香肠表面的油污，将其自然悬挂在恒温培养箱内，于35℃恒温发酵16h；在107cfu混合菌液中，***克鲁维酵母∶鼠李糖乳杆菌＝1.0∶2.5；
(5)干燥后获得成品：
发酵后的香肠置于50℃干燥箱，干燥至含水量≤25％，即为成品发酵血肠。
实施例3：用鸡肉和鸡血进行具有高抗氧化活性的发酵血肠制作方法，包括以下步骤：
(1)Plastein反应制备血红蛋白抗氧化肽：
收集新鲜鸡血，将占新鲜鸡血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，将抗凝血再4000r/min离心15min，收集沉淀的血细胞，取沉淀的血细胞溶于体积比为0.6倍的蒸馏水中，经40kHz超声破碎10min后，调节pH值至2.5作为血细胞反应底物；按重量比，血细胞反应底物∶胃蛋白酶＝3000∶1添加胃蛋白酶，并在37℃下酶解7.5h；经4000r/min离心15min后，取上清为血细胞肽液；按血细胞肽液重量的1％加入L-脯氨酸(Pro)和1％加入L-组氨酸(His)，调节pH值至7.5，30℃保温5.0h后，冷却至室温，真空浓缩至固形物含量为50％左右，获得鸡血红蛋白抗氧化肽备用；胃蛋白酶活力为10万U/g；
(2)畜禽肉腌制和一次发酵处理：
将去掉脂肪、筋膜、肌腱的新鲜牦牛肉，切成5cm见方的肉块，按照重量比，每100份肉加入葡萄糖2.50份、食盐2.50份、五香粉0.28份、味精0.08份、焦磷酸钠0.25份，搅拌均匀，放于4℃腌制10h；
将活化好的肉葡萄球菌(Staphylococcus carnosus)菌液按每克新鲜畜牦牛肉放入106cfu与腌制肉块混匀，密封后在32-37℃发酵24h；获得一次发酵猪肉备用；
(3)畜禽血脱腥处理及破碎：
收集新鲜鸡血，将占新鲜鸡血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，调节pH至5.0-5.5，将抗凝血体积比0.1％的酸性脂肪酶加入抗凝血，于37℃酶解30min，再95℃灭酶活性5min，获得脱腥血液；因热处理后脱腥血液已凝固，需将脱腥血液破碎成2.5-3.0cm见方的脱腥血液小颗粒备用；酸性脂肪酶活力为10万U/g；
(4)灌制及二次发酵：
将重量比血红蛋白抗氧化肽∶一次发酵畜禽瘦肉∶脱腥血液小颗粒＝0.2∶1.0∶4.0混合成为肽肉血混物，按每克肽肉血混物放入107cfu***克鲁维酵母(Kluyveromycesmarxianus)和鼠李糖乳杆菌(Lactobacillus rhamnosus)的混合菌液，并搅拌均匀，最后灌入天然的猪小肠肠衣，用温水洗净香肠表面的油污，将其自然悬挂在恒温培养箱内，于35℃恒温发酵186h；在107cfu混合菌液中，***克鲁维酵母∶鼠李糖乳杆菌＝1.0∶2.5；
(5)干燥后获得成品：
发酵后的香肠置于50℃干燥箱，干燥至含水量≤25％，即为成品发酵血肠。

Claims (1)

1.一种具有高抗氧化活性的发酵血肠制作方法，其特征在于，包括以下步骤：
(1)Plastein反应制备血红蛋白抗氧化肽：
收集新鲜畜禽血，将占新鲜畜禽血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，将抗凝血再4000r/min离心15min，收集沉淀的血细胞，取沉淀的血细胞溶于重量比为0.6-0.8倍的蒸馏水中，经40kHz超声破碎10min后，调节pH值至2.5作为血细胞反应底物；按重量比，血细胞反应底物∶胃蛋白酶＝3000∶1添加胃蛋白酶，并在37℃下酶解4.0-7.5h；经4000r/min离心15min后，取上清为血细胞肽液；按血细胞肽液重量的1％加入L-脯氨酸(Pro)和1％加入L-组氨酸(His)，调节pH值至7.5，30℃保温4.0-6.0h后，冷却至室温，真空浓缩至固形物含量为50％左右，获得血红蛋白抗氧化肽备用；胃蛋白酶活力为10万U/g；
(2)畜禽肉腌制和一次发酵处理：
将去掉脂肪、筋膜、肌腱的新鲜畜禽瘦肉，切成5cm见方的肉块，按照重量比，每100份肉加入葡萄糖2.00-3.00份、食盐3.00-4.00份、五香粉0.12-0.25份、味精0.05-0.08份、焦磷酸钠0.20-0.40份，搅拌均匀，放于4℃腌制8-12h；
将活化好的肉葡萄球菌(Staphylococcus carnosus)菌液按每克新鲜畜禽瘦肉放入106cfu与腌制肉块混匀，密封后在32-37℃发酵24h；获得一次发酵畜禽瘦肉备用；
(3)畜禽血脱腥处理及破碎：
收集新鲜畜禽血，将占新鲜畜禽血重量比10％的柠檬酸钠加入到新鲜畜禽血中溶化后迅速抗凝，获得抗凝血，调节pH至5.0-5.5，将抗凝血体积比0.1％的酸性脂肪酶加入抗凝血，于37℃酶解30min，再95℃灭酶活性5min，获得脱腥血液；因热处理后脱腥血液已凝固，需将脱腥血液破碎成2.5-3.0cm见方的脱腥血液小颗粒备用；酸性脂肪酶活力为10万U/g；
(4)灌制及二次发酵：
将重量比血红蛋白抗氧化肽∶一次发酵畜禽瘦肉∶脱腥血液小颗粒＝0.2∶1.0∶4.0混合成为肽肉血混物，按每克肽肉血混物放入107cfu***克鲁维酵母(Kluyveromycesmarxianus)和鼠李糖乳杆菌(Lactobacillus rhamnosus)的混合菌液，并搅拌均匀，最后灌入天然的猪小肠肠衣，用温水洗净香肠表面的油污，将其自然悬挂在恒温培养箱内，于35℃恒温发酵18h；在107cfu混合菌液中，***克鲁维酵母∶鼠李糖乳杆菌＝1.0∶2.5；
(5)干燥后获得成品：
发酵后的香肠置于50℃干燥箱，干燥至含水量≤25％，即为成品发酵血肠。
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