CN116406781A - 发酵牡蛎调味品及其制备方法与应用 - Google Patents
发酵牡蛎调味品及其制备方法与应用 Download PDFInfo
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- CN116406781A CN116406781A CN202310213676.8A CN202310213676A CN116406781A CN 116406781 A CN116406781 A CN 116406781A CN 202310213676 A CN202310213676 A CN 202310213676A CN 116406781 A CN116406781 A CN 116406781A
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- oyster
- enzymolysis
- fermented
- seasoning
- fermentation
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Abstract
本发明公开了一种发酵牡蛎调味品及其制备方法与应用,属于发酵产品技术领域。所述发酵牡蛎调味品通过以下方法制备得到:(1)牡蛎脱盐;(2)牡蛎匀浆与加水均质;(3)酶解;(4)钝酶;(5)发酵基质的制备:向牡蛎酶解液中添加酪蛋白、乳糖和氯化钠,混匀,灭菌,得发酵基质;(6)发酵:将复合菌种接入发酵基质中,30℃条件下密封发酵24~48h,即得发酵牡蛎调味品;所述复合菌种是由戊糖乳杆菌、植物乳杆菌和酿酒酵母组成的。所述发酵牡蛎调味品在作为食品添加剂中的应用。本发明的发酵牡蛎调味品,保持了全牡蛎的营养价值,呈味氨基酸及游离脂肪酸酸含量高,丰富了酯类风味物质的种类和含量,实现了牡蛎基料的整体风味改善。
Description
技术领域
本发明涉及一种发酵牡蛎调味品及其制备方法与应用,属于发酵产品技术领域。
背景技术
牡蛎是经济上重要的海洋贝类。太平洋牡蛎(Crassostrea gigas)是中国山东的主要养殖品种。它含有优质蛋白质、维生素和矿物质,尤其是牛磺酸。牡蛎的传统加工方法是腌制、煮沸、烘烤和油炸。高温煮沸是开发基础材料风味的最广泛使用的方法。蚝汁经牡蛎加工熬制浓缩形成,不仅能增香提味,也有丰富的生物活性物质。但是其加工时间长,产率低,生产成本较高。
蛋白酶水解是实现牡蛎全利用及调味品基料开发的主要方式。已有的研究表明,牡蛎水解物具有血管紧张素转换酶抑制、抗疲劳和壮阳活性等。然而,加工方法的不同导致了产品质量不均匀和营养损失。外肽酶从蛋白质的C或N末端起作用,而内肽酶通过靶向肽键产生肽和游离氨基酸(FAA),从而极大地改变产品的风味。牡蛎水解物通常表现出强烈的异味,如鱼腥味,限制了其进一步加工和产品开发。如何有效提高牡蛎水解物的风味是牡蛎高值化利用及产业升级所面临的一个重要问题。
乳酸菌和酵母菌作为重要的食品微生物,在中国传统的发酵食品中的应用已有数千年的历史,被广泛用于多种发酵食品(如乳制品、肉类、酸面团、蔬菜等)的发酵剂。在发酵过程中,它们通过改善发酵食品的维生素、氨基酸、生物活性肽或化学成分的风味、味道、质地和生物利用度做出贡献。微生物酶对可发酵碳水化合物、含氮化合物和其他食物基质起作用,同时在发酵过程中将底物转化为有机酸、氨基酸、生物活性化合物和挥发性成分。食品基质和负责发酵生态中风味的代谢物的化学成分也经历了动态变化过程。因此,这些具有优良发酵特性的微生物可以分离、筛选并用作食品生产的发酵剂。
发明内容
针对上述现有技术,为实现牡蛎的综合利用和深加工,本发明提供了一种发酵牡蛎调味品及其制备方法与应用。本发明的发酵牡蛎调味品具有高附加值和良好的风味特性,可作为食品添加剂用于提升风味。
本发明是通过以下技术方案实现的:
一种发酵牡蛎调味品的制备方法,包括以下步骤:
(1)牡蛎脱盐:取牡蛎,去壳,牡蛎肉在冰水浸泡条件下浸泡脱盐;
(2)牡蛎匀浆与加水均质:脱盐后的牡蛎肉进行匀浆,然后加入适量水,均质;
(3)酶解:加入蛋白酶进行酶解,得牡蛎酶解液;
所述蛋白酶选自现有技术中已有的用于水解动物蛋白的蛋白酶;所述酶解的酶解条件(包括酶的添加量、酶解pH、温度、时间)可参考商品说明书,通常情况下,酶解温度为50~55℃,酶解时间为4~5h;
(4)钝酶:酶解后,牡蛎酶解液在超高压450~550Mpa条件下保压20min进行钝酶,或水浴煮沸10min进行钝酶;
(5)发酵基质的制备:钝酶后,向牡蛎酶解液中添加酪蛋白、乳糖和氯化钠,混匀,灭菌(121℃灭菌30min),得发酵基质;所述酪蛋白、乳糖和氯化钠的添加量分别为牡蛎酶解物的5%~10%、3%~5%和1%~3%,质量体积比,单位g/mL;
(6)发酵:将复合菌种接入发酵基质中,接种量4%~8%(g/mL),优选6%;置于恒温培养箱中,30℃条件下密封发酵24~48h,即得发酵牡蛎调味品;
所述复合菌种是由戊糖乳杆菌(Lactobacilluspentosus)、植物乳杆菌(Lactobacillus plantarum)和酿酒酵母(Saccharomyces cerevisiae)组成的,其中,戊糖乳杆菌、植物乳杆菌、酿酒酵母三者的比例(重量比)为(1~2):(0.8~1.2):(0.8~1.2)。
进一步地,所述步骤(2)中,牡蛎肉与水的重量比为1:1~4,优选1:2,1:3;
进一步地,所述步骤(3)中,蛋白酶选自南宁东恒华道生物科技有限责任公司生产的动物蛋白水解专用复配蛋白酶(无商品号);所述酶解的酶解条件为:加酶量为3000U/g,初始pH值为6.5,酶解温度为50℃,酶解时间为4h。
进一步地,所述步骤(5)中,酪蛋白是通过以下方法制备得到的:取牛乳,以及醋酸-醋酸钠缓冲液(pH 4.7),分别预加热至40℃,在搅拌条件下缓慢地将二者混合得悬乳液(二者体积比1:1);冷却至室温,离心(3000r/min,10min),弃去上清液,得酪蛋白粗制品;水洗1~3次,离心,弃去上清液,向沉淀中加入适量乙醇,搅拌均匀,布氏漏斗抽滤,用乙醇-***混合液(1:1,v/v)洗沉淀2次,最后用***洗沉淀2次,干燥,即得酪蛋白。
利用上述方法制备得到的发酵牡蛎调味品,可作为食品添加剂用于提升风味。
本发明采用特定的复合菌种对牡蛎进行发酵,菌种(戊糖乳杆菌、植物乳杆菌和酿酒酵母)均分离于自发酵的牡蛎(原料自适性强),具有降低不饱和醛酮类化合物含量的潜力,且能够解决牡蛎酶解液的腥味和苦味问题。本发明的发酵牡蛎调味品,保持了全牡蛎的营养价值,呈味氨基酸及游离脂肪酸酸含量高,丰富了酯类风味物质的种类和含量,实现了牡蛎基料的整体风味改善。本发明为牡蛎基调味品的开发利用奠定了基础。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。
附图说明
图1:菌种的表面疏水性和自动聚集性示意图,a)为表面疏水性,b)为自动聚集性。
图2:菌种的胃肠适应性示意图。
图3:不同方法制备的牡蛎调味品的氨基酸组成示意图。
图4:不同方法制备的牡蛎调味品的脂肪酸组成示意图。
图5:不同方法制备的牡蛎调味品的分子感官评价示意图,其中,a)为描述词得分雷达图,b)为总体得分。
图6:不同方法制备的牡蛎调味品的的风味物质组成示意图1。
图7:不同方法制备的牡蛎调味品的的风味物质组成示意图2。
图8:不同方法制备的牡蛎调味品的的风味物质组成示意图3。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域技术人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料,可通过正规商业途径获得。下述实施例中所涉及的实验方法、检测方法等,若无特别说明,均为现有技术中已有的常规实验方法、检测方法。
实施例1制备牡蛎发酵基质
步骤如下:
(1)牡蛎脱盐:去壳的牡蛎500g,在4℃、2L冰水浸泡条件下浸泡30min脱盐,沥干水分后再加入2L冰水重复浸泡30min;重复4次;
(2)牡蛎匀浆与加水均质:脱盐后牡蛎肉进行匀浆,匀浆的转速为4500rpm,匀浆15min;按照料液比1:2的比例添加蒸馏水,使用均质分散机均质分散,均质分散机转速为5000rpm,均质时间8min;
(3)酶解:均质分散后的混合液的pH为6.42,加入0.2%复配蛋白酶,于恒温摇床中进行酶解,酶活力为50000U/g,酶解温度为50℃,酶解时间为4h;所使用的复配蛋白酶是南宁东恒华道生物科技有限责任公司生产的动物蛋白水解专用复配蛋白酶;
(4)钝酶:酶解后,牡蛎酶解液在超高压450MPa的条件下保压20min进行钝酶;
(5)酪蛋白的制备:取100mL牛乳、100mL醋酸-醋酸钠缓冲液(pH 4.7),分别预加热至40℃,在搅拌条件下缓慢地将二者混合;悬乳液冷却至室温,离心(3000r/min,10min),弃去上清液,得酪蛋白粗制品;用少量水洗沉淀3次,重复离心,弃去上清液;向沉淀中加入30mL乙醇,搅拌均匀,转移至布氏漏斗中抽滤,用乙醇-***混合液洗沉淀2次,用***洗沉淀2次,干燥,得酪蛋白纯品;
(6)发酵基质的制备:钝酶后,向牡蛎酶解液中添加酪蛋白、乳糖和氯化钠,酪蛋白、乳糖和氯化钠的添加量分别为牡蛎酶解物的8%、3%和2%;混匀,121℃灭菌30min,得发酵基质,发酵基质均一稳定,固形物含量为15.6%,pH为6.07。
实施例2制备牡蛎发酵基质
步骤如下:
(1)牡蛎脱盐:去壳的牡蛎500g,在4℃、2L冰水浸泡条件下浸泡30min脱盐,沥干水分后再加入2L冰水重复浸泡30min;重复4次;
(2)牡蛎匀浆与加水均质:脱盐后牡蛎肉进行匀浆,匀浆的转速为3000rpm,匀浆15min;按照料液比1:3的比例添加蒸馏水,使用均质分散机均质分散,均质分散机转速为5000rpm,均质时间8min;
(3)酶解:均质分散后的混合液的pH为6.86,加入0.2%复配蛋白酶,于恒温摇床中进行酶解,酶活力为50000U/g,酶解温度为50℃,酶解时间为4h;所使用的复配蛋白酶是南宁东恒华道生物科技有限责任公司生产的动物蛋白水解专用复配蛋白酶;
(4)钝酶:酶解后,牡蛎酶解液水浴煮沸10min进行钝酶;
(5)酪蛋白的制备:取100mL牛乳、100mL醋酸-醋酸钠缓冲液(pH 4.7),分别预加热至40℃,在搅拌条件下缓慢地将二者混合;悬乳液冷却至室温,离心(3000r/min,10min),弃去上清液,得酪蛋白粗制品;用少量水洗沉淀3次,重复离心,弃去上清液;向沉淀中加入30mL乙醇,搅拌均匀,转移至布氏漏斗中抽滤,用乙醇-***混合液洗沉淀2次,用***洗沉淀2次,干燥,得酪蛋白纯品;
(6)发酵基质的制备:钝酶后,向牡蛎酶解液中添加酪蛋白、乳糖和氯化钠,酪蛋白、乳糖和氯化钠的添加量分别为牡蛎酶解物的6%、4%和1%;混匀,121℃灭菌30min,得发酵基质,发酵基质均一稳定,固形物含量为12.8%,pH为6.47。
实施例3制备牡蛎发酵基质
步骤如下:
(1)牡蛎脱盐:去壳的牡蛎500g,在4℃、2L冰水浸泡条件下浸泡30min脱盐,沥干水分后再加入2L冰水重复浸泡30min;重复4次;
(2)牡蛎匀浆与加水均质:脱盐后牡蛎肉进行匀浆,匀浆的转速为4500rpm,匀浆15min;按照料液比1:3的比例添加蒸馏水,使用均质分散机均质分散,均质分散机转速为5000rpm,均质时间8min;
(3)酶解:均质分散后的混合液的pH为6.51,加入0.2%复配蛋白酶,于恒温摇床中进行酶解,得牡蛎酶解液,酶活力为50000U/g,酶解温度为50℃,酶解时间为4h;所使用的复配蛋白酶是南宁东恒华道生物科技有限责任公司生产的动物蛋白水解专用复配蛋白酶;
(4)钝酶:酶解后,牡蛎酶解液在超高压550MPa的条件下保压20min进行钝酶;
(5)酪蛋白的制备:取100mL牛乳、100mL醋酸-醋酸钠缓冲液(pH 4.7),分别预加热至40℃,在搅拌条件下缓慢地将二者混合;悬乳液冷却至室温,离心(3000r/min,10min),弃去上清液,得酪蛋白粗制品;用少量水洗沉淀3次,重复离心,弃去上清液;向沉淀中加入30mL乙醇,搅拌均匀,转移至布氏漏斗中抽滤,用乙醇-***混合液洗沉淀2次,用***洗沉淀2次,干燥,得酪蛋白纯品;
(6)发酵基质的制备:钝酶后,向牡蛎酶解液中添加酪蛋白、乳糖和氯化钠,酪蛋白、乳糖和氯化钠的添加量分别为牡蛎酶解物的8%、5%和1%;混匀,121℃灭菌30min,得发酵基质,发酵基质均一稳定,固形物含量为16.1%,pH为5.82。
上述牡蛎匀浆、牡蛎酶解液、发酵基质中多肽的分子量统计如表1所示。
由表1可见,牡蛎酶解液中80%以上为1kDa以下的小分子多肽,经菌种发酵后有70%以上的小分子肽存在于发酵基质中,因此发酵基质具有丰富的营养价值和生物活性,能够实现牡蛎的全利用,保持了牡蛎本身的风味。
表1不同牡蛎样品的分子量分布
实施例4菌种筛选
本发明从发酵牡蛎中筛选得到了三个性能优异的菌种,经鉴定,分别为:戊糖乳杆菌(Lactobacilluspentosus)、植物乳杆菌(Lactobacillusplantarum)和酿酒酵母(Saccharomyces cerevisiae),其表面疏水性如图1a)所示,其自动聚集性如图1b)所示,其胃肠适应性如图2所示。
由图1可知,三种菌种表面疏水性最好的菌株为戊糖乳杆菌,对二甲苯的疏水率为57.62±2.3%。自动聚集能力是指菌体在培养过程中,大量并快速地繁殖从而导致其自行聚集成团的现象,戊糖乳杆菌的聚集性为50.14±1.2%。说明其中戊糖乳杆菌的肠道粘附性较好,菌体对肠上皮细胞的黏附性对其功能特性具有重要的作用。
如果菌体对人或动物消化道中存在或分泌的各种消化酶(主要是胃蛋白酶、胰酶等)或胃液和肠液具有良好的耐受能力,乳酸菌进入人或动物体内后,则可在消化道中顺利存活并定殖,从而持久地发挥其益生功能,有效预防宿主胃肠道疾病的发生。由图2可知,戊糖乳杆菌和植物乳杆菌在模拟胃肠道转运后活菌数最高,分别为6.36Log CFU/g和5.84LogCFU/g。酿酒酵母的活菌数为4.28Log CFU/g。乳酸菌和酵母菌在人或动物的胃肠道中能有较高的存活率对其在体内的定殖和其益生功能的发挥十分重要,这对乳酸菌类的益生菌乳制品或其他益生产品的开发和生产具有重要意义。因此,这三种菌都可用于牡蛎制品的发酵调味品开发。本发明所涉及的这三个菌种均为常规菌种,技术人员可常规从发酵牡蛎中筛选得到,也可市场购买得到。
实施例5制备发酵牡蛎调味品
将复合菌种(由戊糖乳杆菌、植物乳杆菌和酿酒酵母组成的,其中,戊糖乳杆菌、植物乳杆菌、酿酒酵母三者的比例为2:1:1)接入实施例3制备的发酵基质中,接种量6%(v/v);置于恒温培养箱中,30℃条件下密封发酵36h,即得发酵牡蛎调味品。
同时,以发酵基质为空白对照,以乳酸菌发酵制备得到的调味品(方法同上,不同之处在于将复合菌种替换为乳酸菌,本发明所用乳酸菌为戊糖乳植物杆菌CICC 22707,购于中国工业微生物菌种保藏管理中心,分离于泡菜)为对照1,以酵母菌发酵制备得到的调味品(方法同上,不同之处在于将复合菌种替换为酵母菌,本发明所用酵母菌为酿酒酵母CICC 1012,购于中国工业微生物菌种保藏管理中心,分离于葡萄酒)为对照2。
空白对照、对照1、对照2、本发明的发酵牡蛎调味品,氨基酸组成如图3所示(图中标注混合菌的,即表示本发明的发酵牡蛎调味品),脂肪酸制成如图4所示。由图3、图4可知,对特征风味影响较大的氨基酸主要有天冬氨酸、苯丙氨酸、蛋氨酸、谷氨酸、亮氨酸、缬氨酸、异亮氨酸和半胱氨酸。对特征风味影响较大的脂肪酸主要有C16:0、C20:5、C20:4、C18:1、C22:6、C18:0和C16:1。牡蛎基质经混合菌发酵后其游离氨基酸总浓度由3239.52mg/100g增加到7035.24mg/100g。牡蛎基质经过酶解加工和SFA含量较高,经菌发酵后,在微生物分解和酶的作用下PUFA含量显著增加,包含脂肪酸,如亚油酸、亚麻酸、硬脂酸和花生四烯酸,在均化后生成各种氢过氧化物,作为风味化合物的重要前体。
分子感官评价:采用风味剖面法筛选感官描述词,“青草味”、“酸味”、“油脂氧化味”、“奶香味”和“腥味”是样品中最主要的风味属性。总体得分以从0(不可感知)到5(极强)的强度等级进行评分。
空白对照、对照1、对照2、本发明的发酵牡蛎调味品,图5a)为描述词得分雷达图,分子感官评价得分如图5b)所示。由图5可知,牡蛎基质的感官特性以腥味和油脂氧化味组成,整体风味较差。经发酵后,调味品的风味属性主要味奶香味、酸味和青草味,风味轮廓发生改变。添加菌种发酵后,与牡蛎基质相比,调味料的整体感官评分显著增加,其中混合菌总体得分为4.63,显著高于其他三组,说明混合菌发酵有效提高了牡蛎调味品的风味。
风味物质组成分析:将牡蛎样品(2mL)分别加入小瓶(20mL)中,并在自动取样器中以250rpm和50℃孵育30分钟。孵育后,将每个牡蛎样品的顶空气体(500μL)注入气相色谱仪,使用MXT-WAX色谱柱(内径15m×0.53mm)。氮气用作载气,具体流程如下:
流速最初为2mL/min,持续2分钟,然后为10mL/min,持续10分钟,100mL/min,持续20min,最后为150mL/min,持续25分钟。对于IMS,漂移管温度为45℃,漂移气体为高纯度氮气,流速恒定(150mL/min)。使用实验室分析查看器软件处理基于分析光谱的指纹图谱。
空白对照、对照1、对照2、本发明的发酵牡蛎调味品,风味物质组成如图6、7、8所示。GC-IMS化合物鉴定表如表2所示。
戊醛、2-甲基-4-庚酮、辛醛、反式-2-(2-戊烯基)呋喃、乙酸、甲酸庚酯、(E,E)-2,4-庚二烯醛、2,3-丁二醇、2-乙基己酸和3-甲基丁酸是发酵牡蛎调味品主要的香气化合物,这些挥发性化合物贡献了酸味、青草味、脂肪味和金属味。
乳酸菌发酵后的牡蛎调味品含有较高的呋喃和烯烃,而酵母菌发酵后以酸类和酯类为主,醛酮类化合物的含量在发酵后明显降低。乳酸乙酯、十六酸乙酯、2-辛烯醛、(E,E)-2,4-癸二烯醛、2-乙酰基呋喃、辛酸乙酯、苯甲醛和异戊酸这些风味物质可作为牡蛎发酵过程中风味品质监测的关键指标。
表2GC-IMS化合物鉴定表
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (10)
1.一种发酵牡蛎调味品的制备方法,其特征在于,包括以下步骤:
(1)牡蛎脱盐:取牡蛎,去壳,牡蛎肉在冰水浸泡条件下浸泡脱盐;
(2)牡蛎匀浆与加水均质:脱盐后的牡蛎肉进行匀浆,然后加入适量水,均质;
(3)酶解:加入蛋白酶进行酶解,得牡蛎酶解液;
(4)钝酶;
(5)发酵基质的制备:钝酶后,向牡蛎酶解液中添加酪蛋白、乳糖和氯化钠,混匀,灭菌,得发酵基质;所述酪蛋白、乳糖和氯化钠的添加量分别为牡蛎酶解物的5%~10%、3%~5%和1%~3%;
(6)发酵:将复合菌种接入发酵基质中,接种量4%~8%,优选6%;置于恒温培养箱中,30℃条件下密封发酵24~48h,即得发酵牡蛎调味品;
所述复合菌种是由戊糖乳杆菌、植物乳杆菌和酿酒酵母组成的,其中,戊糖乳杆菌、植物乳杆菌、酿酒酵母三者的比例为(1~2):(0.8~1.2):(0.8~1.2)。
2.根据权利要求1所述的发酵牡蛎调味品的制备方法,其特征在于:所述步骤(2)中,牡蛎肉与水的重量比为1:1~4。
3.根据权利要求1所述的发酵牡蛎调味品的制备方法,其特征在于:所述步骤(3)中,酶解的酶解条件为:酶解温度50~55℃,酶解时间4~5h。
4.根据权利要求1所述的发酵牡蛎调味品的制备方法,其特征在于:所述步骤(3)中,蛋白酶选自南宁东恒华道生物科技有限责任公司生产的动物蛋白水解专用复配蛋白酶;所述酶解的酶解条件为:加酶量为3000U/g,初始pH值为6.5,酶解温度50℃,酶解时间4h。
5.根据权利要求1所述的发酵牡蛎调味品的制备方法,其特征在于:所述步骤(4)中,钝酶的具体操作为:酶解后,牡蛎酶解液在超高压450~550Mpa条件下保压20min进行钝酶,或水浴煮沸10min进行钝酶。
6.根据权利要求1所述的发酵牡蛎调味品的制备方法,其特征在于:所述步骤(5)中,酪蛋白是通过以下方法制备得到的:取牛乳,以及醋酸-醋酸钠缓冲液,分别预加热至40℃,在搅拌条件下将二者混合得悬乳液;冷却至室温,离心,弃去上清液,得酪蛋白粗制品;水洗1~3次,离心,弃去上清液,向沉淀中加入适量乙醇,搅拌均匀,布氏漏斗抽滤,用乙醇-***混合液洗沉淀2次,最后用***洗沉淀2次,干燥,即得酪蛋白。
7.根据权利要求1所述的发酵牡蛎调味品的制备方法,其特征在于:所述步骤(5)中,酪蛋白、乳糖和氯化钠的添加量分别为牡蛎酶解物的8%、3%和2%;或:6%、4%和1%;或:8%、5%和1%。
8.根据权利要求1所述的发酵牡蛎调味品的制备方法,其特征在于:所述步骤(6)中,复合菌种中,戊糖乳杆菌、植物乳杆菌、酿酒酵母三者的比例为2:1:1;接种量为6%。
9.利用权利要求1~8中任一项所述的方法制备得到的发酵牡蛎调味品。
10.权利要求9所述的发酵牡蛎调味品在作为食品添加剂中的应用。
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