CN114874914A - 一株广谱抑菌自絮凝非酿酒酵母菌株cc-p5及其应用 - Google Patents
一株广谱抑菌自絮凝非酿酒酵母菌株cc-p5及其应用 Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明公开了非酿酒酵母Hanseniaspora vineae菌株CC‑P5，它的保藏编号为CGMCC No.23571。所述的非酿酒酵母Hanseniaspora vineae菌株CC‑P5在生物防控中的应用；所述的生物防控是在微生物发酵过程中能防止杂菌的污染。该菌株来源安全，分离于葡萄果实表面。该株菌具有良好的抑菌作用；在发酵的过程中具有很强的絮凝能力（絮凝率89.41%）且具有生物防控能力，不仅在发酵的过程中不易污染杂菌，而且在发酵完成后无须离心，通过自沉降既能达到分离菌体的目的，具有良好的应用价值。
Description
技术领域
本发明属于微生物发酵技术领域，具体涉及一株广谱抑菌自絮凝非酿酒酵母菌株CC-P5及其应用。
背景技术
非酿酒酵母由于其在发酵的过程中具有积极的作用，越来越受到人们的关注。非酿酒酵母可以产生甘油、高级醇及其它挥发性化合物，有利于提升葡萄酒的香气、风味等感官特征。许多具有有益特性的非酿酒酵母有助于增加葡萄酒的风味和提高葡萄酒的质量。非酿酒酵母可以分为3组：（1）好氧型酵母，例如毕赤酵母属、德巴利酵母属、红酵母属等；（2）低发酵活性的酵母，例如有孢汉逊酵母、罕见有孢汉逊酵母；（3）高发酵活性酵母，例如马克斯克鲁维酵母等。
自絮凝酵母在发酵的过程中有很多优点，无须离心能够快速从发酵液中沉降分离，提高了生产效率减少了能耗，而且收集的菌体可以重复使用。同时，发酵过程中杂菌的污染和致病菌的繁殖都会影响到发酵产品的最终质量和安全性，造成经济损失。因此具有生物防控能力的自絮凝菌株在工业上具有良好的应用价值。
发明内容
本发明目的是提供一株广谱抑菌自絮凝非酿酒酵母菌株CC-P5及其应用。
非酿酒酵母Hanseniaspora vineae菌株CC-P5，它的保藏编号为CGMCC No.23571。
所述的非酿酒酵母Hanseniaspora vineae菌株CC-P5在抑制Escherichia coli O157:H7、methicillin-resistant Staphylococcus aureus (ATCC43300)、Staphylococcus aureus (ATCC 29213)、Salmonella choleraesuis (ATCC 50019)、 Riemerella anatipestifer (ATCC 11845)、Escherichia coli (CICC 10899)、Listeria monocytogenes (ATCC 19111)的应用；
所述的生物防控是在微生物发酵过程中能防止杂菌的污染。
本发明提供了非酿酒酵母Hanseniaspora vineae菌株CC-P5，它的保藏编号为CGMCC No.23571。所述的非酿酒酵母Hanseniaspora vineae菌株CC-P5在生物防控中的应用；所述的生物防控是在微生物发酵过程中能防止杂菌的污染。该菌株来源安全，分离于葡萄果实表面。该株菌对Escherichia coli O157:H7、methicillin-resistant Staphylococcus aureus (ATCC43300)、Staphylococcus aureus (ATCC 29213)、 Salmonella choleraesuis (ATCC 50019)、Riemerella anatipestifer (ATCC 11845)、 Escherichia coli (CICC 10899)、Listeria monocytogenes (ATCC 19111)具有良好的抑菌作用。该菌株在发酵的过程中具有很强的絮凝能力（絮凝率89.41%）且具有生物防控能力，不仅在发酵的过程中不易污染杂菌，而且在发酵完成后无须离心，通过自沉降既能达到分离菌体的目的，具有良好的应用价值。
附图说明
图1为Hanseniaspora vineae菌株CC-P5结晶紫染色后的形态；
图2为Hanseniaspora vineae菌株CC-P5在YPD液体培养基中的自絮凝情况，所示为涡旋1min后的状态；
图3为Hanseniasporavineae菌株CC-P5的抑菌谱。
具体实施方式
实施例1非酿酒酵母Hanseniaspora vineae菌株CC-P5的分离及鉴定
1、分离纯化：从水果店购买葡萄后，半小时内运回实验室；取葡萄果实表皮，放置无菌袋中并添加适量的无菌PBS，进行摇晃清洗，制成原液。从原液中吸取1mL进行梯度稀释，分别取100μL的10-1、10-2、10-3和10-4的稀释液涂布在含有25μg/mL氯霉素的YPD琼脂平板上。将平板在30℃培养3-7天后，挑取平板上的可疑菌落进行分离纯化。
2、分子鉴定：提取已纯化酵母的DNA作为模板，对26S D1/D2和ITS区域进行PCR扩增测序，所用引物为NL1 (5′-GCA TAT CAA TAAGCG GAG GAA AAG-3′)/NL4 (5′-GGT CCGTGTTTC AAG ACG G-3′)和ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′)/ITS4 (5′-TCC TCCGCT TAT TGA TAT GC-3′)。将PCR产物测序之后，经BLAST比对，其26S D1/D2和ITS区域与Hanseniasporavineae菌株的相似性都达99 %以上，因此可认为该菌株为葡萄酒有孢汉逊酵母Hanseniasporavineae菌株，命名为Hanseniaspora vineaeCC-P5。该菌株已于2021年10月11日保藏在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏编号为CGMCCNo.23571。
实施例2 菌株的自絮凝能力测定
挑取Hanseniaspora vineae菌株CC-P5于5ml YPD液体培养基中，30℃，120rpm培养48h后，涡旋10s静置10min再小心吸取上层200μL菌液测量OD600数值，记为OD initial；再将菌液3000rpm离心5min，收集菌体并用PBS进行清洗，重新悬浮在PBS中并添加适量的木瓜蛋白酶对菌体进行解絮，完全解絮后，3000rpm离心5min收集菌体并用PBS进行清洗，最后将菌液重悬在5mLYPD培养基中并吸取上层200μL菌液测量OD600数值，记为ODafter；其絮凝率（%）=(1- ODinitial/ODafter)×100。经计算该菌絮凝率89.41%。
实施例3 菌株抑菌谱
挑取Hanseniaspora vineae菌株CC-P5于5mlYPD液体培养基中，30℃，48h后，3000rpm离心5min收集菌体，并根据实施例2所述对菌体进行解絮，解絮之后吸取10μL的菌液在YPD琼脂板中心划线，30℃下培养72h。将用TSB培养基培养好的指示菌（Escherichia coli O157:H7、methicillin-resistant Staphylococcus aureus (ATCC43300)、Staphylococcus aureus(ATCC 29213)、Salmonella choleraesuis (ATCC 50019)、 Riemerella anatipestifer(ATCC 11845)、 Escherichia coli (CICC 10899)、Listeria monocytogenes(ATCC 19111)），调节至菌落浓度约为1×106 cfu/mL，分别取5μL在培养72h后的YPD琼脂板上垂直Hanseniaspora vineae CC-P5进行划线，划线完成后，将平板在37℃下培养20-24h，观察Hanseniaspora vineae CC-P5对指示菌的抑菌作用。根据图3可知，该菌对Escherichia coli O157:H7、methicillin-resistant Staphylococcus aureus (ATCC43300)、Staphylococcus aureus(ATCC 29213)、Salmonella choleraesuis (ATCC50019)、Riemerella anatipestifer(ATCC 11845)、 Escherichia coli (CICC 10899)、 Listeria monocytogenes(ATCC 19111)具有抑菌作用。
Claims (3)
1.非酿酒酵母Hanseniaspora vineae菌株CC-P5，它的保藏编号为CGMCC No.23571。
2.权利要求1所述的非酿酒酵母Hanseniaspora vineae菌株CC-P5在抑制Escherichia coli O157:H7、methicillin-resistant Staphylococcus aureus (ATCC43300)、Staphylococcus aureus ATCC 29213、Salmonella choleraesuis ATCC 50019、 Riemerella anatipestifer ATCC 11845、Escherichia coli CICC 10899、Listeria monocytogenes ATCC 19111的应用。
3.根据权利要求2所述的应用，其特征在于：所述的生物防控是在微生物发酵过程中能防止杂菌的污染。
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