CN116286441B - 一株具有降解生物胺特性的腐生葡萄球菌及其在哈尔滨风干肠中的应用 - Google Patents
一株具有降解生物胺特性的腐生葡萄球菌及其在哈尔滨风干肠中的应用 Download PDFInfo
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Abstract
本发明公开了一株具有降生物作用腐生葡萄球菌及其应用，属于微生物技术领域。本发明的菌株命名为腐生葡萄球菌JX9(Staphylococcus saprophyticus JX9)，保藏于中国微生物菌株保藏管理委员会普通微生物中心，保藏号为：CGMCC No.25286。通过在培养基中对4种不产生物胺的细菌进行降生物胺能力测定，发现4株菌株均有不同程度的降胺能力，且腐生葡萄球菌JX9在多种生物胺的降解中表现突出；进而对这4株菌株进行胺氧化酶活力测定，结果表明腐生葡萄球菌JX9的胺氧化酶活性最高。将其接种到哈尔滨风干肠中，发现腐生葡萄球菌JX9风干肠中的腐胺、尸胺、组胺、酪胺、亚精胺和精胺含量显著低与未接种的对照组，降解率分别为75.04％、86.67％、100％、75.21％、60.67％和56.51％，且总生物胺降解率达69.37％。因此，该菌株的发现为降低发酵肉制品中的生物胺含量提供了有效的解决办法。
Description
技术领域
本发明属于微生物技术领域，涉及一株具有降解生物胺特性的腐生葡萄球菌JX9及其在哈尔滨风干肠中的应用。
背景技术
生物胺是广泛存在于发酵食品中的一类具有生物活性的含氮有机碱，它主要通过酶促氨基酸脱羧以及酮和醛的还原性胺化而形成。根据其结构通常可分为脂肪族胺(腐胺、尸胺、胍胺、精胺、亚精胺)、芳香族胺(色胺、β-苯乙胺)和杂环胺(组胺和色胺)。机体中少量的生物胺能发挥积极的作用，如作为合成激素、核酸、蛋白质的前体，调节体温、调节胃容积和pH等；过量的生物胺则会危害人体健康，导致血压升高、严重头痛、高血压危象、出血和心力衰竭。
胺氧化酶是一类能够催化胺类氧化脱氨的特殊蛋白质，可作用于单胺、二胺与多胺的伯胺基和仲胺基，将其氧化生成相应的醛、氨和过氧化氢。根据氧化酶含有的辅基不同，可分为含铜胺氧化酶和含黄素胺氧化酶，其中含铜胺氧化酶包括二胺氧化酶(DAO)和伯胺氧化酶(PrAO)，含黄素胺氧化酶包括单胺氧化酶(MAO)和多胺氧化酶(PAO)。虽然生物胺能通常通过小肠中的单胺氧化酶(MAO)和二胺氧化酶(DAO)被分解，但一旦饮酒或食用了单胺氧化酶抑制剂生物胺的作用可能会被大幅度增强。此外，在亚硝酸盐存在的情况下，生物胺也可能转化为潜在致癌的N-亚硝胺，导致产品品质下降。特别是发酵肉制品，在其发酵过程中的pH逐渐下降，微生物为抵抗酸性条件胁迫作用更易通过脱羧作用产生生物胺。因此，筛选具有胺能降解生物胺并有胺氧化酶活性的菌株并接种于发酵食品，在其制作过程中从源头抑制生物胺的生成或对其进行降解显得尤为重要。
凝固酶阴性葡萄球菌是风干肠发酵过程中广泛应用的发酵剂之一。它能够在风干肠的发酵中代谢碳水化合物、发生酯化反应和分解脂肪等使风干肠具有独特的风味，且葡萄球菌能够将脂肪和蛋白质降解为游离氨基酸和脂肪酸，有利于机体对这些营养物质的吸收。有研究表明，葡萄球菌能够通过产生氧化酶进而促进食品中生物胺的降解。生物胺被降解生成小分子低毒性产物，在细胞正常代谢中被吸收排出，从而完成降解。此外，微生物所产生的细胞代谢物也可能抑制产生物胺菌株脱羧酶活性，进而控制生物胺的形成。因此，通过采用凝固酶阴性葡萄球菌作为风干肠的发酵剂以降解生物胺是一种安全、有效的方式。
发明内容
本发明的目的是公开一株具有降生物胺能力的凝固酶阴性葡萄球菌，并应用于哈尔滨风干肠中，以控制哈尔滨发酵风干肠中的生物胺含量。
(1)本发明的腐生葡萄球菌JX9由由东北农业大学食品学院自传统发酵风干肠中分离得到，保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号中科院微生物研究所，其菌种保藏编号为：CGMCC No.25286，保藏日期为2022年7月13日。
(2)本发明的腐生葡萄球菌JX9在培养基中的生物胺降解模拟试验中呈现良好的降生物胺能力，其对色胺、苯乙胺、腐胺、尸胺、组胺、酪胺、精胺和亚精胺的降解率分别为17.00％、17.42％、15.78％、16.71％、10.06％、31.72％、25.05％和22.03％；胞外单胺氧化酶活性、胞内单胺氧化酶活性、胞外二胺氧化酶活性和胞内二胺氧化酶活性分别为117.76、1361.50、86.43和81.48U/g蛋白；经接种至风干肠中验证，其在风干肠中对腐胺、尸胺、组胺、酪胺、精胺和亚精胺的均有显著的降解效果(P<0.05)，且总生物胺的降解率达56.43％。因此，腐生葡萄球菌JX9可以作为有降生物胺的功能性发酵剂应用到发酵肉制品中，以生产低生物胺发酵肉制品。
附图说明
图1为4株试验菌株在培养基中的生物胺降解模拟试验中对八种生物胺的降解率；
图2为4株试验菌株的生物胺氧化酶活力测定结果；
图3为风干肠中接种腐生葡萄球菌JX9后八种生物胺含量变化。
具体实施方式
下面结合附图对本发明的技术方案作进一步的说明，但并不局限于此，凡是对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，均应涵盖在本发明的保护范围中。
一.培养基中腐生葡萄球菌对生物胺降解能力的测定
(1)MRA培养基的配制
大豆蛋白胨3.5g，酵母浸粉6.5g，酪蛋白胨10.0g，甘露醇5.0g，甘氨酸10.0g，磷酸氢二钾5.0g，氯化锂5.0g，丙酮酸钠10.0g，蒸馏水1000mL，pH 7.2～7.4。
(2)生物胺降解率的测定
将分离自风干肠中的菌株经溴甲酚紫显色反应试验获得4株不产生物胺细菌(腐生葡萄球菌SYS1、干酪巨大球菌SYS4、表皮葡萄球菌JX6和腐生葡萄球菌)，进行生物胺降解能力测定。4株试验菌株分别接种至MRA培养基中活化3代后，分别以体积比为2％接种量接种至MRA-BA培养基(在MRA培养基中分别加入50mg/L的色胺、苯乙胺、组胺、酪胺、腐胺、尸胺、亚精胺和精胺)，以不接种菌株的培养基为对照，32℃培养4天。将20种菌液于4℃、6000r/min下离心10min，取0.2mL离心后的菌液于试管中，加入0.8mL 20％TCA溶液提取，遵循GB 5009.208—2016《食品中生物胺的测定》第一法液相色谱法中的酒类及调味品(醋和酱油)方法进行生物胺含量测定。
生物胺降解率的计算方法：
式中：C0为对照组培养基中的生物胺浓度(mg/L)；C1为20株试验菌株培养基中的生物胺浓度(mg/L)
二.培养基中葡萄球菌胺氧化酶活力测定
将这4株细菌进行单胺氧化酶(MAO)和二胺氧化(DAO)活性检测。4株菌株在MRA培养基中活化3代后，于4℃、6000r/min下离心10min，上清液以70％硫酸铵盐析提取蛋白，弃去上清液后将得到的蛋白于等体积PBS中溶解，测定蛋白浓度后于4℃待用，用于测定胞外氧化酶活力；离心后的菌泥用PBS清洗2次后重悬于等体积PBS，加入蛋白酶抑制剂苯甲基磺酰氟0.02mmol/L，冰浴下在超声破碎仪上将细胞打碎，2s开，2s关，交替破碎循环共15min。将破碎的细胞在4℃、10000r/min下离心15min，收集上清液测定蛋白浓度后于4℃待用，用于测定胞内胺氧化酶活力。蛋白含量测定采用BCA蛋白含量测定试剂盒(上海碧云天生物技术有限公司)；单胺氧化酶活力测定采用单胺氧化酶试剂盒(南京建成生物工程研究所)；二胺氧化酶活力测定采用二胺氧化酶试剂盒(南京建成生物工程研究所)。
三.风干肠中生物胺降解能力验证
(1)风干肠的制备
原料：瘦肉(猪里脊)4.5kg，肥肉(猪背脂)0.5kg，大麯酒0.15kg，盐0.125kg，亚硝酸钠0.0045kg，绵白糖0.25kg，味素0.075kg，混合调料(世一堂干肠料)0.025kg。
发酵剂制备：将四株生物胺降解能力较强的葡萄球菌在MSA培养基中进行两次传代后，于32℃培养18h。4℃、10,000g离心5min后，备用。添加发酵剂时，葡萄球菌添加量为107cfu/g肉馅。
工艺流程：原料肉(剔除淋巴，筋腱，血管等结缔组织)→切丁(1cm3的肉丁)→腌制(加盐和调味料等)→加发酵剂(对照组不添加)→拌馅→灌制(长度20cm，直径3cm)→发酵风干(温度25±2℃，湿度65±5％)。
(2)风干肠中生物胺的测定
遵循GB 5009.208—2016《食品中生物胺的测定》第一法液相色谱法中水产品和肉类的测定方法进行风干肠中生物胺含量的测定。
四.结果与分析
(1)从图1可知，4株葡萄球菌均能不同程度降解生物胺，各生物胺的降解率在10.00-32.97％，其中腐生葡萄球菌JX9在色胺、苯乙胺、酪胺、亚精胺和精胺的降解上表现突出，说明该菌株具有较强的降低多种生物胺的能力。
(2)进一步对4株试验菌株进行胞内与胞外胺氧化酶活力测定，如图2所示。结果表明，4株试验菌株的胞内和胞外均有单胺和二胺氧化酶活力，其中腐生葡萄球菌的胞内与胞外胺氧化酶活力显著高于其他3株菌株(P<0.05),表明该菌株具备更强的生物胺降解潜力。
(3)图3表明，腐生葡萄球菌JX9接种至风干肠中能有效抑制风干肠中的生物胺含量，其中对腐胺、尸胺、组胺、酪胺、精胺和亚精胺含量的抑制效果显著(P<0.05)，降解率分别为75.04％、86.67％、100％、75.21％、60.67％和56.51％，且总生物胺的降解率达69.37％。试验结果表明，腐生葡萄球菌JX9具有显著降低生物胺的能力，能够应用于发酵肉制品的生产中以降低产品的生物胺含量。
Claims (2)
1.一株具有降生物胺作用的腐生葡萄球菌(Staphylococcus saprophyticus)JX9，保藏于中国微生物菌种保藏管理委员会普通微生物中心，其菌种保藏编号为：CGMCCNo.25286。
2.如权利要求1所述的腐生葡萄球菌JX9在哈尔滨风干肠中降低生物胺含量的应用。
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