CN116410874A - 一株发酵毕赤酵母及其在制备发酵酸肉中的应用 - Google Patents
一株发酵毕赤酵母及其在制备发酵酸肉中的应用 Download PDFInfo
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- CN116410874A CN116410874A CN202210989715.9A CN202210989715A CN116410874A CN 116410874 A CN116410874 A CN 116410874A CN 202210989715 A CN202210989715 A CN 202210989715A CN 116410874 A CN116410874 A CN 116410874A
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- pichia
- pichia pastoris
- fermented
- meat
- fermentans
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Abstract
本发明属于微生物技术领域，涉及发酵酸肉用微生物，具体涉及一株毕赤酵母及其在制备发酵酸肉中的应用。本发明公开了一株发酵毕赤酵母(Pichia fermentans)ZY‑1，保藏号为CGMCC NO.24770，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日期为2022年4月26日，本发明的发酵毕赤酵母(Pichia fermentans)ZY‑1能显著增加具有果香味的酯类、青草味的醛类物质的相对含量，改善并丰富了发酵酸肉的风味特征，将其作为发酵剂应用于其他发酵肉制品具有广大的应用前景。
Description
技术领域
本发明属于微生物技术领域，涉及发酵酸肉用微生物，具体涉及一株发酵毕赤酵母及其在制备发酵酸肉中的应用。
背景技术
发酵酸肉是一种传统自然发酵的肉制品，经过发酵，肉中的蛋白质、脂肪等物质在微生物及酶的作用下产生氨基酸、脂肪酸等风味前体物质，赋予产品独特风味和营养；另一方面，肉中大量有益微生物在发酵过程中生长繁殖，成为发酵肉制品中的优势微生物，使发酵肉制品中腐败微生物的生长繁殖及亚硝酸盐等有害物质的产生受到限制，较大程度地提高了发酵肉制品的安全性。
发酵酸肉中较高的盐浓度(低水活度)与低pH等环境条件，有利于酵母菌的生长繁殖。酵母菌能通过糖酵解、蛋白水解和脂肪分解，促进酸肉中酯类、酮类、醇类等挥发性风味物质的产生。还可通过降解过氧化物和消耗氧气来延缓酸败并保护红色亚硝基肌红蛋白免受分解，从而稳定酸肉的色泽，改善其感官特性。
目前国内关于以本土酵母菌作为发酵剂应用于发酵肉中的研究较少，而商业化的发酵剂对于产品风味的贡献并不突出。酵母产香的研究主要集中于白酒类食品，而应用于发酵酸肉制品上的研究鲜有报道。从产品中筛选到的产香酵母不仅具有较强的生长代谢能力，同时其在生长繁殖过程中能促进酯类、醛类等大量挥发性物质的释放。因此，利用酵母菌这一特征，将其作为发酵剂接种至肉中发酵一定时间，对于改善并提高发酵酸肉品质稳定性、赋予其独特风味特征具有重要意义。
发明内容
本发明的目的就是根据现有技术中存在的问题，提供一种能产生挥发性风味物质的毕赤酵母及其在肉品发酵中的应用。
本发明通过如下技术方案实现：
一种能产生挥发性风味物质的发酵毕赤酵母(Pichia fermentans)ZY-1，该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24770，保藏日期为2022年4月26日，保藏地址：北京市朝阳区北辰西路1号院3号，其5.8SrRNA核苷酸序列为如SEQ ID NO.1所示。
本发明提供的发酵毕赤酵母(Pichia fermentans)ZY-1是从发酵酸肉样品中分离得到的。
本发明提供的发酵毕赤酵母(Pichia fermentans)ZY-1在PDA培养基上生长良好，菌落形态为乳白色，扁平，不透明，表面皱缩无光泽，边缘不整齐，细胞形态呈椭圆形。
本发明提供的发酵毕赤酵母(Pichia fermentans)ZY-1，通过提取总DNA、PCR扩增后，将得到的基因序列与NCBI在线基因数据库中的已知序列进行比对，得到其与已知标准菌株，编号为KM029994.1的Pichia fermentans HR-1DS 1308相似度为99.75％。
本发明提供的发酵毕赤酵母(Pichia fermentans)ZY-1应用于发酵酸肉中，固相微萃取结合气相色谱质谱联用仪测得接种了发酵毕赤酵母(Pichia fermentans)ZY-1的酸肉中，共得到38种挥发性物质：25种酯类、5种酸类、2种醇类、5种醛类、1种酮类，关键挥发性风味物质(ROAV大于1)有丁酸乙酯、庚酸乙酯、辛酸乙酯、癸酸乙酯、正己酸乙酯、2-甲基丁酸丙酯、戊酸乙酯、正己醇、1-辛烯-3-醇、癸醛、苯乙醛、壬醛等9种。与未接种毕赤酵母的酸肉相比，接种发酵毕赤酵母(Pichia fermentans)ZY-1能使酸肉中酯类物质的相对含量由36.83％显著增加至41.33％(p＜0.01)，醛类物质的相对含量由0.77％显著增加至1.93％(p＜0.01)。
本发明提供的发酵毕赤酵母(Pichia fermentans)ZY-1能显著增加具有果香味的酯类、青草味的醛类物质的相对含量，改善并丰富了发酵酸肉的风味特征，将其作为发酵剂应用于其他发酵肉制品具有广大的应用前景。
附图说明
图1为本发明发酵毕赤酵母(Pichia fermentans)ZY-1在PDA培养基上的菌落形态图；
图2为本发明发酵毕赤酵母(Pichia fermentans)ZY-1的细胞形态图；
图3为本发明发酵毕赤酵母(Pichia fermentans)ZY-1鉴定的系统发育树图；
图4为本发明发酵毕赤酵母(Pichia fermentans)ZY-1强化发酵酸肉后挥发性物质的种类数及相对含量图；
图5为本发明发酵毕赤酵母(Pichia fermentans)ZY-1强化发酵酸肉后全部挥发性物质组成热图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：菌株的分离、筛选
称取发酵酸肉样品25.0g，添加至225mL无菌生理盐水中，于28℃，150rpm的摇床振荡30分钟。然后再梯度稀释，分别得到10-2、10-3、10-4的菌悬液。移液枪吸取10-1-10-4的菌悬液0.2mL分别涂布在马铃薯葡萄糖琼脂培养基(PDA)上，每个稀释度涂布两个平板。将涂布好的平板置于28℃的恒温培养箱中培养5天后，从10-1-10-4稀释度的平板上各挑取15-20株不同形态但具有毕赤酵母典型菌落特征的菌株，进一步划线分离纯化。重复2-3次，结合显微镜观察菌落是否纯化。根据其在固体培养基上的菌落形态并结合嗅闻培养皿上的气味，从发酵酸肉样品中分离纯化到的菌株中筛选出目标发酵毕赤酵母(Pichia fermentans)ZY-1，观察该菌株的菌落形态及细胞形态特征。挑取纯化好的单个菌落于酵母膏胨葡萄糖(YPD)琼脂培养基上划线，然后将斜面培养基置于28℃培养箱中培养，菌落长满斜面后，将培养基置于4℃冰箱中保藏。如图1、2所示，发酵毕赤酵母(Pichia fermentans)ZY-1在PDA培养基上的菌落形态及其显微镜下的细胞形态如附图1和图2所示：菌落呈乳白色，扁平，不透明，表面皱缩无光泽，边缘不整齐，细胞形态为椭圆形，繁殖方式为出芽生殖。
实施例2菌株的鉴定
准确吸取1mL发酵毕赤酵母(Pichia fermentans)ZY-1的菌悬液于平板中，分别倒入碳源、氮源同化培养基，再添加浓度50mmol/L的过0.22μm滤膜的碳源(葡萄糖、乳糖、蔗糖、麦芽糖、柠檬酸)和氮源(硝酸钾、硫酸铵)于培养基表面，30℃培养24小时，以无碳(氮)源培养基为对照实验，观察培养基上各菌落生长情况。
碳源同化培养基：硫酸铵5g；磷酸氢钾1g；七水硫酸镁0.5g；酵母浸膏0.2g；琼脂20g；水1000mL，灭菌条件：115℃，15min。
氮源同化培养基：葡萄糖20g；磷酸二氢钾1g；七水硫酸镁0.5g；酵母浸膏0.2g；琼脂20g；水1000mL；pH：6.4-6.6，灭菌条件：115℃，15min。
发酵毕赤酵母(Pichia fermentans)ZY-1的碳源、氮源同化试验结果如表1所示，发酵毕赤酵母(Pichia fermentans)ZY-1可利用葡萄糖、柠檬酸为碳源，硝酸钾为氮源。
表1发酵毕赤酵母碳源、氮源同化试验结果
| 化合物 | Z | 化合物 | Z | 化合物 | Z |
| 葡萄糖 | + | 乳糖 | - | 麦芽糖 | - |
| 化合物 | Z | 化合物 | Z | 化合物 | Z |
| 蔗糖 | - | 柠檬酸 | + | 纤维二糖 | - |
| 化合物 | Z | 化合物 | Z | ||
| 硫酸铵 | - | 硝酸钾 | + |
注：+表示反应为阳性，-表示反应为阴性。
DNA提取试剂盒提取发酵毕赤酵母(Pichia fermentans)ZY-1的总DNA后，使用真菌通用引物ITS1/ITS4对其进行PCR扩增。将PCR扩增产物进行测序，得到发酵毕赤酵母(Pichia fermentans)ZY-1的5.8SrRNA核苷酸序列如SEQ ID NO.1所示。NCBI网站将获得的发酵毕赤酵母(Pichia fermentans)ZY-1的基因序列进行BLAST(Basic Local AlignmentSearch Tool)，选取数据库中与测定序列同源性高于99％的序列进行比对，比对结果显示，发酵毕赤酵母(Pichia fermentans)ZY-1与与已知标准菌株，编号为KM029994.1的Pichiafermentans HR-1 DS 1308相似度为99.75％。进一步将得到的比对结果使用MEGA软件中的Neighbor-joining算法构建系统发育树(附图3)，将目标菌株鉴定为发酵毕赤酵母(Pichiafermentans)。该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24770，保藏日期为2022年4月26日。
实施例3菌株的应用
1、挑取试管中的发酵毕赤酵母(Pichia fermentans)ZY-1在PDA培养基上划线，于28℃培养5天以活化菌株。将活化好的发酵毕赤酵母(Pichia fermentans)ZY-1作为发酵剂(菌液浓度1.0×107cfu/g)按照原料肉的1％添加至肉中，以不添加发酵剂的酸肉作为空白对照组，置于通风良好的室内，自然发酵30天。发酵结束后，GC-MS测定两组酸肉中的挥发性风味物质，得到发酵毕赤酵母(Pichia fermentans)ZY-1强化发酵后对酸肉整体风味的影响。
2、酸肉中挥发性风味物质的测定(SPME-GC-MS)：
①.SPME条件：称取3g样品于10mL萃取瓶后，将萃取头插入萃取瓶置于磁力搅拌器(温度设置为60℃)上离样品1cm的位置平衡5min后推出纤维头，提取样品中的挥发性成分，时间为40分钟；挥发性成分萃取完成后，250℃条件下于进样口以无分裂模式解吸5min，启动气相色谱质谱仪检测。
②.GC条件：以HP-5MS毛细管柱(30m×0.25mm，0.25μm)为色谱柱，进样方式：不分流，以纯度≥99.999％的氦气作为载气，进样口温度250℃，流速控制在1.0mL/min；柱温设置如下：50℃保持1min，8℃/min升至130℃，保持1min，最后以15℃/min升至280℃，保持2min；后运行程序：280℃、3min。
③.MS条件：电子能量70eV，离子源温度230℃，m/z为30～450，电离方式：电子电离源。
④.挥发性成分的定性及定量：将所识别到的挥发性物质质谱中的离子碎片、质荷比等相关参数进行比对，得到与NIST17.L质谱数据库匹配度大于80％的化合物进行分析；酸肉发酵过程中检测到的各挥发性成分的相对含量(％)通过峰面积归一化法实现。根据挥发性物质的相对含量及其对应的感觉阈值得到相对气味活度值(ROAV)，当ROAV≥1时被认为该物质对被测样品的整体风味有贡献。
3、结果与分析
GC-MS从对照组及发酵毕赤酵母(Pichia fermentans)ZY-1发酵酸肉样品中均检测到38种挥发性物质。其中酯类物质种数最多，相对含量最高，分别为25种，41.39％；酸类物质5种，相对含量为2.95％；醇类物质为2种，相对含量为2.19％；醛类物质5种，相对含量为1.93％；酮类物质1种，相对含量为0.23％。如附图4所示，与未接种菌株的酸肉(NF组)相比，发酵毕赤酵母(Pichia fermentans)ZY-1强化发酵(PF组)能够极显著增加酯类物质和醛类物质的相对含量(p＜0.01)，极显著降低酸类和酮类物质的相对含量(p＜0.01)，而对醇类无显著性作用。酯类物质及醛类物质是发酵酸肉中的主要挥发性物质，能够赋予酸肉水果香及青草香气。毕赤酵母能够促进酯化反应的进行，因此酸类物质的相对含量显著降低和醇类物质相对含量无显著性降低。检测到的接种与未接种发酵毕赤酵母(Pichiafermentans)ZY-1酸肉中全部挥发性物质的相对含量及相对气味活度值(ROAV)见表2。由此可知，酸肉中含量丰富的酯类、酸类、醇类及醛类物质，赋予了产品独特的风味特征。
表2样品中各挥发性物质的相对含量及相对气味活度值(ROAV)
注：“/”表示未检测到
发酵毕赤酵母(Pichia fermentans)ZY-1强化发酵30天后，产生了16种以酯类物质为主的新的挥发性物质：甲酸异戊酯、油酸乙酯、甲酸辛酯、巴豆酸乙烯酯、S-甲基2-甲基硫代丙酸酯、9-十六碳烯酸乙酯、乙醇酸乙酯、甲酸己酯、戊酸、2-甲基丁酸、2-甲基戊酸、1-辛烯-3-醇、癸醛、2-苯基-2-丁烯醛、十六醛、4-甲基-1,2,4-三唑烷-3,5-二酮。同时显著增加了4-甲基戊酸异戊酯、5-甲基己酸乙酯、辛酸乙酯、月桂酸乙酯、癸酸乙酯、正己酸乙酯、3-苯丙酸乙酯、棕榈酸乙酯、4-甲基戊酸、苯乙醛、壬醛等11种挥发性物质的相对含量。其中1-辛烯-3-醇、苯乙醛、壬醛、癸酸乙酯、正己酸乙酯的相对气味活度值大于1，对酸肉整体风味具有重要贡献作用。正己酸乙酯的相对含量达20.71％，呈菠萝果香；癸酸乙酯具有微弱果香味，类似坚果香气，含量为0.39％；具有花香的苯乙醛相对含量为1.53％；壬醛相对含量为0.3％。具有玫瑰柑橘香和油脂气味；1-辛烯-3-醇具有蘑菇香，相对含量为0.18％。通过标准化的热图更直观地展示各挥发性物质在两组酸肉中的分布，颜色越深代表该挥发性物质在酸肉中的相对含量越高，由附图5可知，发酵毕赤酵母(Pichia fermentans)ZY-1显著影响了酸肉中挥发性物质的组成。
接种发酵毕赤酵母(Pichia fermentans)ZY-1引起的这些差异挥发性物质，使得发酵酸肉的风味特征更丰富、更独特，将其作为产香发酵剂应用具有广阔的应用前景，对产品良好品质的形成具有重要意义。
上述内容仅为本发明的实施例，但本发明的范围不局限于此。应当指出，在不偏离本范围技术范围的前提下，可以对本发明的技术方案进行改进，改进后的内容同为本发明的保护范围。
Claims (7)
1.一株发酵毕赤酵母(Pichia fermentans)ZY-1，其特征在于，所述毕赤酵母(Pichiafermentans)ZY-1的保藏号为CGMCC NO.24770，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日期为2022年4月26日。
2.一种发酵剂，其特征在于，所述发酵剂中含有权利要求1所述的发酵毕赤酵母(Pichia fermentans)ZY-1。
3.根据权利要求2所述的发酵剂，其特征在于，所述发酵毕赤酵母(Pichiafermentans)ZY-1的菌液浓度为1.0×107cfu/g。
4.权利要求1所述的发酵毕赤酵母(Pichia fermentans)ZY-1或权利要求2或3所述的发酵剂在制备发酵酸肉中的应用。
5.一种发酵酸肉的制备方法，其特征在于，所述方法包括以下步骤：
(1)将权利要求1所述的发酵毕赤酵母(Pichia fermentans)ZY-1进行活化，得发酵剂；
(2)将步骤(1)所述的发酵剂接种于原料肉中发酵得到所述的发酵酸肉。
6.根据权利要求5所述的制备方法，其特征在于，所述发酵剂中发酵毕赤酵母(Pichiafermentans)ZY-1的菌液浓度1.0×107cfu/g。
7.根据权利要求6所述的制备方方法，其特征在于，所述步骤(2)中发酵剂按照原料肉的1％添加于原料肉中，自然发酵30天。
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