CN117660197A - 一种米曲霉及其应用 - Google Patents
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Abstract
本发明提供一种米曲霉及其应用,属于微生物技术领域。本发明的米曲霉命名为米曲霉(Aspergillusoryzae)ZA198,已于2021年5月18日保藏于广东省微生物菌种保藏中心,地址:广州市先烈中路100号大院59号楼5楼广东省微生物研究所,保藏编号为GDMCC No.61676。将本发明的米曲霉(Aspergillusoryzae)ZA198应用于酱油或酱生产中,无需额外添加微生物和调配剂能够有效改善酱油或酱的风味,降低酿造过程产生的生青味等杂味物质,同时能够提升酱油或酱的品质,使酱油或酱的口感风味更加纯净。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种米曲霉及其应用。
背景技术
传统发酵食品是指以动植物为原料,通过天然微生物或人工添加微生物的一系列反应生产出的能满足公众需求的美味、营养的产品。其中发酵豆制品中的酱油是一种以大豆、小麦等粮食作物为主要原料,经米曲霉等微生物发酵酿造而成具有独特色、香、味的传统调味品,其同时混合了鲜味、甜味、酸味、酯香味和酱香味等多种风味,受到广大消费者的青睐。酱油中风味物质非常复杂,不是单一的物质,而是几十种甚至上百种复杂化合物的混合物。这些香气化合物包括醇、醛、酸、酯、酮、呋喃、酚、硫化物等。酱油不同风味、香气的产生和形成主要来源于它不同的生产酿造工艺及原料、配方、原料处理和生产过程中微生物作用的好坏。在酿造过程中由于微生物的作用使之产生一系列的生化反应把原料中的不溶性高分子物质分解成低分子化合物。这些分解物的相互组合、多级转化和微生物的自溶作用生成种类繁多的呈味生香营养物。这些物质相辅相成就构成了酱油的特殊风味和香气,然而,由于微生物及各种条件的不可控性,发酵过程中往往会伴随杂味和不良风味的出现,当其中的某些具有不良风味的物质超过一定剂量时,便会产生异味。如己醛会产生辛辣的青草气味,反-2-辛烯醛会产生生青味,1-辛烯-3-酮会产生草本味和土味,1-辛烯-3-醇是一类酱油曲霉孢子的特征风味,具有蘑菇特征风味,主要来源于原料的制曲阶段,浓度过高会掩盖酱油原本酱香风味。
调控各种风味成分的种类、含量是优化酱油风味的有效手段,目前改善酱油风味的途径有选择适宜的原料和适当的原料配比、适量添加微生物、通过调配改善风味。其中额外添加微生物改善酱油风味需要考虑不同微生物之间的最佳培养与发酵条件,以及在发酵过程中如何去调控对原有发酵体系的影响等问题,会导致额外的生产工序和生产成本的增加。通过添加其他物质对酱油进行调配改善酱油风味需要考虑对酱油色泽、感官、稳定性等的影响,同时也不符合消费者追求天然无添加产品的健康饮食心理。因此有必要开发出一株降低不良风味和杂味提升酱油纯净味品质的米曲霉菌株。中国发明专利202110976111.6公开一株米曲霉ZA256及其应用,用该菌株制备酱油,所得酱油1-辛烯-3-醇含量高,是1-辛烯-3-醇相对丰度高且相对峰面积大的高品质酱油,克服了传统酱油香气格局单一的缺陷。中国发明专利202110732532.4公开一株米曲霉ZA223及其应用,用该菌株制备酱油具备醇类、酯类物质含量高,醛类、酮类、酸类物质含量低的特点,赋予了酱油原油果香味,可用于生产新型高端果香味酱油。中国发明专利202110710124.9公开一种提升酱油风味的方法,利用成曲液态自身酶解为原料,先后接种米曲霉和鲁氏酵母获得4-羟基-2(5)-乙基-5(2)-甲基-3(2H)呋喃酮(HEMF)产生的前体和酶,然后添加到酱醪中促进酱油发酵提高HEMF含量进而提升酱油风味。然而,上述专利中提及的酱油风味变化均不涉及降低生青味等杂味物质。
因此,开发一株降低不良风味和杂味,提升酿造酱油品质的米曲霉菌株在酱及酱油酿造具有非常大的生产应用价值。
发明内容
为了解决上述技术问题,本发明提供一种米曲霉及其应用,本发明诱变得到的米曲霉能有效降低酿造酱油和酱的不良风味和杂味,提高酱油纯净品质,使其感官风味更加纯正。
为实现上述目的,本发明提供一种米曲霉,所述米曲霉命名为米曲霉(Aspergillusoryzae)ZA198,已于2021年5月18日保藏于广东省微生物菌种保藏中心,地址:广州市先烈中路100号大院59号楼5楼广东省微生物研究所,保藏编号为GDMCCNo.61676。
为降低传统发酵产品不良风味,提高其优良风味和品质,针对酱油酿造过程产生的己醛、反-2-辛烯醛、1-辛烯-3-酮、1-辛烯-3-醇等杂味物质,无需额外添加微生物和调配剂,本发明采用ARTP诱变方法对出发菌株米曲霉As3.951进行诱变获得突变菌落,经三角瓶培养筛选、成曲制备复筛和酱油原油发酵验证,检测制曲成曲指标和发酵酱油质量及感官评定,获得一株低生青味米曲霉诱变菌株,能够有效降低发酵酱油中己醛、反-2-辛烯醛、1-辛烯-3-酮、1-辛烯-3-醇含量。
本发明还提供所述的米曲霉(Aspergillusoryzae)ZA198在制曲中的应用。
采用本发明的米曲霉(Aspergillusoryzae)ZA198制曲所得成曲的曲香纯正,生青味低并且中性蛋白酶和谷氨酰胺酶活力略高。
作为本发明所述米曲霉的优选实施方式,所述制曲获得的成曲用于制备酱油或酱。
本发明还提供一种成曲,通过将权利要求1所述的米曲霉ZA198(Aspergillusoryzae)接种于大豆原料和/或小麦原料中培养得到。
本发明还提供所述的米曲霉(Aspergillusoryzae)ZA198在制备调味品中的应用。
作为本发明所述应用的优选实施方式,所述调味品包括酱油或酱。
本发明还提供所述的米曲霉(Aspergillus oryzae)ZA198在降低酱油或酱中的杂味物质含量中的应用。
作为本发明所述应用的优选实施方式,所述杂味物质包括己醛、1-辛烯-3-酮、反-2-辛烯醛、1-辛烯-3-醇中的至少一种。
采用本发明的米曲霉菌株发酵酱油或酱,能够降低酱油及酱醅中己醛、反-2-辛烯醛、1-辛烯-3-酮和1-辛烯-3-醇含量,获得口感更加鲜甜、感官风味更加纯净的优良酱油及酱醅。
本发明还提供一种酱油或酱的制备方法,采用如权利要求1所述的米曲霉(Aspergillus oryzae)ZA198作为发酵菌,或采用所述的成曲进行发酵。
本发明还提供一种酱油或酱,采用所述酱油或酱的制备方法制备得到。
与现有技术相比,本发明的有益效果为:本发明提供一种米曲霉及其应用,本发明以米曲霉As3.951为出发菌株,采用公知诱变方法(优选为ARTP诱变)对出发菌株进行诱变获得米曲霉(Aspergillusoryzae)ZA198;本发明的米曲霉(Aspergillusoryzae)ZA198具有菌丝略长、菌丝层略厚、产孢略少、成曲孢子略少、PH高且曲香味淡等特征,特别是使发酵酱油香气物质组成中己醛、反-2-辛烯醛、1-辛烯-3-酮、1-辛烯-3-醇等杂味物质的含量低等特点;将本发明的米曲霉(Aspergillusoryzae)ZA198应用于酱油或酱生产中,无需额外添加微生物和调配剂能够有效改善酱油或酱的风味,降低酿造过程产生的生青味等杂味物质,同时能够提升酱油或酱的品质,使酱油或酱的口感风味更加纯净。
附图说明
图1为本发明实施例米曲霉(Aspergillusoryzae)ZA198的诱变与筛选技术路线图。
图2为米曲霉(Aspergillusoryzae)ZA198在豆汁培养基上的菌落形态。
图3为实施例3发酵酱香气强度鉴评分析。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,本发明通过下列实施例进一步说明。显然,下列实施例仅是本发明的一部分实施例,而不是全部的实施例。应理解,本发明实施例仅用于说明本发明的技术效果,而非用于限制本发明的保护范围。除非特别说明,否则本发明实施例中采用的方法和试验条件均为本领域常规使用的方法和试验条件,使用的试剂、设备和培养基均为本领域常规使用的试剂、设备和培养基,均可通过现有方法制备获得或通过市售购买获得。
实施例1菌株的诱变与筛选
本发明的菌株诱变与筛选的技术路线如图1所示。
1、菌种诱变:
(1)取沪酿As3.951菌株的成熟斜面孢子制成孢子悬液,并对孢子悬液进行ARTP诱变(功率设置为120W,气量10SLM,处理时间控制在0s~300s但不含有0s)。用0.85%的无菌生理盐水将诱变后的孢子悬液逐级稀释到10-1、10-2、10-3、10-4、10-5、10-6浓度,制成孢子稀释液。
(2)取0.2mL孢子稀释液分别涂布于酪素平板培养基和察氏琼脂培养基平板上,31℃避光培养96h。培养过程中观察并记录菌落的菌丝生长和产孢情况、透明圈形成的快慢及透明圈和菌落直径大小、孢子色泽等。挑选与出发菌种As3.951相比具有菌丝略长、菌丝层略厚、产孢子量一般略少特点的菌落。
(3)将上述两种培养基平板上挑选出的目标菌落继续涂布于豆汁培养基平板上,31℃培养68h。培养过程中观察并记录菌落的菌丝生长和产孢情况等。挑选与出发菌种As3.951相比具有菌丝略长、菌丝层略厚、产孢子量一般略少且孢子细的菌落。
(4)选取满足以上平板筛选条件的突变菌株转接豆汁培养基斜面上,培养成熟后进行常规保藏及进一步筛选,菌株编号为YA101~YA105。
其中,所使用的察氏培养基为商业培养基。酪素平板培养基的配方为:干酪素6g,KH2PO4 0.36g,Na2HPO4 0.58g,MgSO4 0.244g,琼脂粉20g,蒸馏水1L,pH6.0,121℃灭菌20min。豆汁平板培养基配方为:KH2PO4 1g,MgSO4 0.5g,(NH4)2SO40.5g,可溶性淀粉20g,琼脂粉20g,豆汁1L,自然pH,121℃灭菌20min。
2、诱变菌株的筛选
(1)诱变菌株的初筛:将步骤1诱变获得的5株目标菌种(YA101~YA105)活化并转接于试管斜面培养基上,培养四天。取成熟斜面培养物分别接种于三角瓶培养基中进行扩培,三角瓶培养基和工艺控制等采用常规方法。其中,培养基是由麸皮、豆粉、面粉、水制备而成。观察并记录培养过程中菌丝生长情况和产孢情况,并进行成曲指标检测,结果如表1所示。挑选菌丝生长略长、菌丝层略厚和孢子着生正常略少的突变菌株YA102、YA103、YA104、YA105进行复筛。
表1不同菌株三角瓶成曲质量分析结果
备注:以对照菌株As3.951各项指标为1.00,其它诱变菌株换算成相应比值
(2)诱变菌株的复筛
将初筛选出的4株菌YA102、YA103、YA104和YA105依次转接斜面活化后,进行三角瓶扩大培养,然后继续进行制曲和发酵小试,制曲培养基采用大豆、小麦等原料制备;培养条件按常规工艺控制。观察并记录培养过程中曲料菌丝生长情况和产孢情况,并进行成曲气味感官和指标检测,检测结果见表2。检测发酵原油中己醛、1-辛烯-3-酮、反-2-辛烯醛和1-辛烯-3-醇的含量,检测结果见表3。
表2不同菌株制曲质量分析结果
备注:以对照菌株As3.951各项理化指标为1.00,其它诱变菌株换算成相应比值。
表3不同菌株发酵原油主要杂味物质含量对比分析结果
根据表2结果可知,相对于出发菌株As3.951,诱变所得菌株YA102~YA105的孢子量略少,成曲pH值略高,中性蛋白酶活力和谷氨酰胺酶活力都略高。根据表3结果可知,相对于出发菌株As3.951,仅有诱变菌株YA105对上述四种挥发性杂味物质均有降低作用,因此优选米曲霉菌株YA105,将上述菌株YA105送进行测序鉴定,鉴定结果为米曲霉(Aspergillusoryzae)。
经上述筛选分离以及鉴定,将菌株YA105进行保藏。发明人已于2021年5月18日保藏于广东省微生物菌种保藏中心,并命名为米曲霉(Aspergillusoryzae)ZA198,保藏编号为GDMCC NO:61676。
本实施例所得的米曲霉(Aspergillusoryzae)ZA198的菌落形态如图2所示。由图2可知,将筛选得到的米曲霉ZA198在豆汁平板培养基培养68h,菌落直径在50mm左右,菌落黄绿色偏绿,菌丝略长、菌丝层略厚、产孢量一般略少。将筛选得到的米曲霉在酪素平板培养基培养96h,菌落直径在23.5mm左右,透明圈直径在28.7mm左右,菌落浅黄绿色,圆形,菌丝生长略长。
实施例2专利菌株米曲霉(Aspergillus oryzae)ZA198发酵酱油效果验证
将诱变筛选的新菌株米曲霉(Aspergillus oryzae)ZA198与出发菌株As3.951同步展开酱油生产使用,制曲结束后抽样检测指标,发酵结束后检测原油指标,并进行原油感官评比。结果如表4、表5、表6和表7所示。
表4制曲质量分析
表5发酵原油质量分析
表6发酵原油主要杂味物质含量对比分析(GC-MS法)
表7发酵原油几种主要香气鉴评结果分析(不同喜好度对比)
由上述可知,将本发明菌株米曲霉(Aspergillus oryzae)ZA198应用于酱油生产,经制曲、发酵压榨后可以提高原油氨基氮、全氮和谷氨酸含量,同时可以使主要杂味物质己醛(辛辣味)、1-辛烯-3-酮(草本味)、反-2-辛烯醛(生青味)和1-辛烯-3-醇(蘑菇味)的含量分别减少13.28%、14%、15.6%和3.98%,使酿造获得的酱油香味更加纯净、风味更加优良,经专业鉴评师评定所得原油香气纯净、杂味弱、酱香纯正,口感绵长无后苦味。
实施例3米曲霉(Aspergillus oryzae)ZA198菌株发酵酱效果验证
将实施例1诱变筛选的新菌株米曲霉(Aspergillus oryzae)ZA198与出发菌株As3.951、米曲霉(Aspergillus oryzae)ZA223(米曲霉ZA223,已于2021年4月30日保藏于广东省微生物菌种保藏中心,保藏号为GDMCC.No:61635,保藏地址为广州市先烈中路100号大院59号楼。)同步展开发酵酱生产使用,并对酱醅中的己醛、1-辛烯-3-酮、反-2-辛烯醛和1-辛烯-3-醇含量进行检测,结果如表8和图3所示。
表8发酵酱醅主要杂味物质含量对比分析(GC-MS法)
由上述可知,将本发明菌株米曲霉(Aspergillus oryzae)ZA198应用于酱醅生产,经发酵后使己醛、反-2-辛烯醛、1-辛烯-3-酮和1-辛烯-3-醇含量分别降低9.39%、4.41%、11.86%和4.44%,减少杂味物质含量,同时经专业鉴评小组评定其发酵出的酱醅香气风味更加纯净。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的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实质和范围。
Claims (10)
1.一种米曲霉,其特征在于,所述米曲霉命名为米曲霉(Aspergillusoryzae)ZA198,已于2021年5月18日保藏于广东省微生物菌种保藏中心,地址:广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No.61676。
2.权利要求1所述的米曲霉(Aspergillusoryzae)ZA198在制曲中的应用。
3.如权利要求2所述的应用,其特征在于,所述制曲获得的成曲用于制备酱油或酱。
4.一种成曲,其特征在于,通过将权利要求1所述的米曲霉ZA198(Aspergillusoryzae)接种于大豆原料和/或小麦原料中培养得到。
5.权利要求1所述的米曲霉(Aspergillusoryzae)ZA198在制备调味品中的应用。
6.根据权利要求5所述的应用,所述调味品包括酱油或酱。
7.权利要求1所述的米曲霉(Aspergillus oryzae)ZA198在降低酱油或酱中的杂味物质含量中的应用。
8.根据权利要求7所述的应用,其特征在于,所述杂味物质包括己醛、1-辛烯-3-酮、反-2-辛烯醛、1-辛烯-3-醇中的至少一种。
9.一种酱油或酱的制备方法,其特征在于,采用如权利要求1所述的米曲霉(Aspergillus oryzae)ZA198作为发酵菌,或采用如权利要求4所述的成曲进行发酵。
10.一种酱油或酱,其特征在于,采用权利要求9所述方法制备得到。
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