CN117004500A - 产核黄素和有机酸的海洋阿萨希丝孢酵母菌株及其应用 - Google Patents
产核黄素和有机酸的海洋阿萨希丝孢酵母菌株及其应用 Download PDFInfo
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- CN117004500A CN117004500A CN202310838593.8A CN202310838593A CN117004500A CN 117004500 A CN117004500 A CN 117004500A CN 202310838593 A CN202310838593 A CN 202310838593A CN 117004500 A CN117004500 A CN 117004500A
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- riboflavin
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Abstract
本发明提供了一株产核黄素和有机酸的阿萨希丝孢酵母菌株(Trichosporon asahii)，所述的海洋阿萨希丝孢酵母命名为YD‑1，保藏于中国典型培养物保藏中心，保藏编号为M20231042，保藏日期为2023年6月19日。所述菌株以葡萄糖和玉米浆为底物发酵生产，得到的发酵液中核黄素产量可高达19.5mg/L；有机酸(苹果酸、柠檬酸与抗坏血酸)产量可达1190.14mg/L，填补了现有技术的空白。此外，所述发酵液的生产方法不仅生产成本低、发酵工艺简便，而且该发酵液的酪氨酸酶抑制率可达76.15％，且有良好的安全性和稳定性，在化妆品领域存在广阔的应用前景，潜在商业价值巨大。
Description
技术领域
本发明属于生物学领域，涉及一株丝孢酵母属菌株；具体涉及一株海洋阿萨希丝孢酵母(Trichosporon asahii)菌株及其在生产富含核黄素和有机酸的发酵液中的用途。
背景技术
随着化妆品科学的发展与“绿色、天然”理念的深入人心，发酵美白类化妆品因其安全性、稳定性与高亲和力，深受消费者喜爱。丝孢酵母属可通过发酵产生B族维生素、维生素C、氨基酸、有机酸、微量元素与麦角甾醇等，化妆品功效应用前景广泛，频繁出现在研究者视角中。Kashiwayama Shinei报道T.kashiwayama的发酵滤液(Pitera)，因其含有丰富的B族维生素、有机酸、微量元素与氨基酸等，可直接添加进化妆品中，具有美白、除皱以及治疗烫伤的功效，即化妆品著名品牌SK-Ⅱ的主要成分。Ines Schulze等从土壤中分离出一株产棕榈烯酸与亚麻酸的T.porosum，而棕榈烯酸与亚麻酸可用作化妆品保湿剂、乳化剂、柔顺剂与抗氧化剂。Irena Kolouchová等发现皮状丝孢酵母(T.cutaneum)同样可以生产棕榈烯酸，将其应用于保湿类化妆品中。HWANG等发现一株产高活性抗真菌合成肽的白吉利丝孢酵母(T.beigelii)，该丝孢酵母同时可以产生B族维生素、维生素C，具有化妆品应用前景。
T.asahii在陆地、水生环境中均有分布，营养型为化能异养；呼吸方式为兼性厌氧型，属于耐碱酵母，但鲜见应用于发酵工业、化妆品工业。林朝栋等对T.asahii菌株进行了研究，通过对其发酵培养基进行优选，制备出了类似于T.kashiwayama菌株的发酵滤液，其可激活抗氧化系统抑制促炎因子的氧化应激，具有较高的抗氧化与抗炎特性。
核黄素是传统化妆品行业中的着色剂、营养强化剂。但研究人员进一步发现，核黄素还存在较好的美白功效。目前，核黄素在化妆品中的应用大多以直接添加或间接递送的方式出现，目前尚未见利用发酵的方法，将其作为发酵产物的一部分应用在化妆品中的报道。有机酸作为化妆品常见功效物质，具有美白肤质、去角质、抗衰老以及补水抗皱的多重功效；但在化妆品的配制中若采用直接添加的方式可能更易引起过敏、发炎等问题。如果采用发酵的方式，使目的菌株产生所需要的有机酸，利用发酵产物相对温和、更亲和人体的优势，同时通过调控发酵工艺，使有机酸以更安全可靠的方式出现在化妆品中。
此外，发明人经过前期研究发现，如果采用发酵策略，将有机酸与核黄素共同通过发酵代谢产生，有望利用二者的协同效应，达到理想的美白功效。但现有技术中，尚未见具备相应功能的丝孢酵母属菌株的报道。
发明内容
本发明提供了一株产核黄素和有机酸的阿萨希丝孢酵母菌株(Trichosporonasahii)，所述菌株以葡萄糖和玉米浆为底物发酵生产，能生产富含核黄素和有机酸的发酵液，不仅生产成本低、发酵工艺简便，可实现大规模工业化生产，而且该发酵液具有较高的酪氨酸酶抑制率，在化妆品领域存在广阔的应用前景。
本发明的技术方案：
一株产核黄素和有机酸的海洋阿萨希丝孢酵母(Trichosporon asahii)，所述的海洋阿萨希丝孢酵母命名为YD-1，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M20231042，保藏日期为2023年6月19日。
所述的海洋阿萨希丝孢酵母YD-1的活化方法，将YD-1菌株接种到YPD固体培养基中，在28-30℃的温度条件下培养1-2天。其中，所述YPD固体培养基的配方为：葡萄糖20g/L，蛋白胨20g/L，酵母提取物10g/L，琼脂20g/L。
一种包含所述的海洋阿萨希丝孢酵母菌菌株YD-1的菌剂。
如前所述的海洋阿萨希丝孢酵母YD-1在发酵生产富含核黄素和有机酸的发酵液中的应用。具体步骤为：将所述的阿萨希丝孢酵母菌菌株YD-1接入种子培养基中进行种子培养，将培养后的种子液接入到发酵培养基，发酵完成后，分离菌体，得到富含核黄素和有机酸的发酵液。其中，所述的种子培养基为葡萄糖20g/L，蛋白胨20g/L，酵母提取物10g/L；所述种子培养的温条件为28℃，培养时间为12-16h。所述的发酵培养基为：葡萄糖35g/L，玉米浆20g/L；所述的发酵条件为：发酵起始pH为3.20，发酵温度为28℃；搅拌速度为200rpm；通气量为1.5m3/h。
本发明的有益效果：
(1)本发明提供了一种可发酵生产富含核黄素和有机酸的发酵液的海洋阿萨希丝孢酵母YD-1，且发酵液中核黄素产量可高达19.5mg/L；有机酸(苹果酸、柠檬酸与抗坏血酸)产量可达1190.14mg/L，填补了现有技术的空白。
(2)采用本发明所述的YD-1菌株生产富含核黄素和有机酸的发酵液时，以葡萄糖和玉米浆为底物进行发酵生产，生产成本低且发酵工艺简单易操作，产业化应用前景广阔。
(3)采用本发明所述的YD-1菌株生产的发酵液，其酪氨酸酶抑制率可达76.15％，且有良好的安全性和稳定性，因此具备应用于化妆品的潜在美白功效和商业价值。
附图说明
附图1为本发明所述的菌株YD-1的系统发育进化树图；
附图2为本发明所述的菌株YD-1的100升发酵罐培养图；
附图3为本发明所述的菌株YD-1的发酵上清滤液酪氨酸酶抑制率图；
附图4为本发明所述的菌株YD-1的发酵液主成分分析(PCA)图；
附图5为本发明所述的菌株YD-1的未发酵组与发酵组的维生素差异热图；
附图6为本发明所述的菌株YD-1的未发酵组与发酵组的有机酸差异热图；
附图7为本发明所述的菌株YD-1的发酵上清滤液长期保藏稳定性图(a)-20℃；(b)4℃；(c)常温；(d)45℃；
附图8为本发明所述的菌株YD-1的上清滤液长期保藏前后核黄素与有机酸含量变化图，不同符号的数据有显著差异(p<0.05)；
附图9为本发明所述的菌株YD-1的发酵上清滤液高温稳定性(70℃)图，不同符号的数据有显著差异(p<0.05)；
附图10为本发明所述的菌株YD-1的发酵上清滤液酸碱稳定性图，不同符号的数据有显著差异(p<0.05)；
附图11为本发明所述的菌株YD-1的发酵上清滤液光照稳定性图，不同符号的数据有显著差异(p<0.05)；
附图12为本发明所述的菌株YD-1的发酵上清滤液对小鼠成纤维细胞(L929细胞)增殖影响图，空白组代表未添加发酵上清滤液的细胞培养基，不同符号的数据有显著差异(p<0.05)；
附图13为本发明所述的菌株YD-1的发酵上清滤液对小鼠黑色素瘤细胞(B16F10细胞)增殖的影响图，空白组代表未添加发酵上清滤液的细胞培养基，不同符号的数据有显著差异(p<0.05)；
附图14为本发明所述的菌株YD-1的不同浓度发酵上清滤液对小鼠体重的影响图；
附图15为本发明所述的菌株YD-1的发酵上清滤液对小鼠黑色素瘤细胞(B16F10细胞)黑色素含量的影响图，不同符号的数据有显著差异(p<0.05)；
附图16为本发明所述的菌株YD-1的发酵上清滤液对小鼠黑色素瘤细胞(B16F10细胞)酪氨酸酶活性影响图，不同符号的数据有显著差异(p<0.05)。
具体实施方式
下面结合实施例对本发明做进一步的说明。
实施例1：菌株的筛选和鉴定
将来自青岛沿海中的浒苔样品，接种到50mL液体YPD培养基中(添加25μg/ml氯霉素)，在28℃，180rpm条件富集培养3天。将获得的上述培养液稀释涂布YPD培养基平板，然后置于28℃培养箱中倒置培养2d。将平板上出现的单菌落，进行分离纯化，并使用核黄素和有机酸筛选培养基(3g/L葡萄糖，5g/L脱脂牛奶，0.5g/L酵母提取物)进行产核黄素和有机酸筛选。发酵在28℃和180rpm条件下振荡培养7天，发酵液用高效液相色谱系统(Agilent1100，安捷伦科技有限公司)测定核黄素和有机酸产量，最终确定YD-1菌株为最优菌株。
使用PCR扩增的方法扩增YD-1菌株基因组26S rDNA序列，引物如表1所示。
表1通用PCR引物
PCR产物经测序后，在NCBI网站上进行26S rDNA序列比对，并选取标准菌株构建26S序列进化树。如图1所示，YD-1菌株种属确定为阿萨希丝孢酵母(Trichosporonasahii)，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M20231042，保藏日期为2023年6月19日。
实施例2：Trichosporon asahii YD-1菌株在100升发酵罐中发酵生产核黄素和有机酸
利用Trichosporon asahii YD-1菌株，在100L机械搅拌发酵罐(BIOTECH-100JS，上海保兴生物设备工程有限公司)中进行核黄素和有机酸的发酵生产。种子培养基为葡萄糖20g/L，蛋白胨20g/L，酵母提取物10g/L，在28℃下培养12-16h，然后以10％接种量接种到发酵培养基中，发酵配培养基配方为葡萄糖35g/L，玉米浆20g/L，控制起始pH 3.2，总体积为70L。发酵温度为28℃，搅拌速度为200rpm，通气量为1.5m3/h。发酵过程中，间隔8h取样一次，采用HPLC检测发酵液中的核黄素与有机酸。结果如图2所示。由图2可知，发酵72h时，核黄素产量可达到19.50mg/L，有机酸产量达到1190.14mg/L。
然而，作为美白化妆品原料的发酵液，需要同时实现核黄素与有机酸的高产量，而发酵48h的发酵液中核黄素含量为16.1±0.32mg/L，有机酸含量为1512.6±0.51mg/L。因此，相较于发酵72h的发酵液来说，发酵48h时的发酵液更符合同时实现核黄素与有机酸高产量的要求。对48h时发酵液进行酪氨酸酶抑制率测定，结果如图3所示。由图3可知，发酵上清滤液的酪氨酸酶抑制率达到76.15±3.50％；阳性对照为100μg/mL的曲酸溶液，其酪氨酸酶抑制率为90.46±4.84％；而空白组的酪氨酸酶抑制率仅为0.50±0.02％。综上所述，发酵48h的发酵液，可以有效抑制调控黑色素生成中关键限速的酶酪氨酸酶的活性，进而减少黑色素的生成，具有一定的美白潜力。
实施例2：Trichosporon asahii YD-1发酵液非靶向代谢组学分析
(1)主成分分析(PCA)
将未接种Trichosporon asahii YD-1菌株的培养基(UF)与100L发酵罐中发酵液(F100L)两组样品进行主成分分析，结果如图4所示。由图4可知，在对样品成分贡献最高的第一主成分维度(PC1，54.44％)上，未发酵组的成分和100L发酵组具有显著差异。而在样品成分贡献次之的第二主成分维度(PC2，28.59％)上，未发酵组与100L发酵组之间亦存在显著的差异。这说明，经过Trichosporon asahii YD-1菌株的发酵，多方面的代谢进程导致发酵液与未发酵的培养基相比，成分已经出现了明显的区别。
(2)不同类别物质差异分析
将未接种Trichosporon asahii YD-1菌株的培养基(UF)、与100L发酵罐中发酵液(F100L)两组样品进行不同类别物质差异分析。维生素在不同阶段相对丰度变化的聚类分析见图5，结果表明，除了硫胺素(维生素B1)在发酵后丰度出现降低，核黄素(维生素B2)、烟酸(维生素B3)、泛酸(维生素B5)抗坏血酸(维生素C)与维生素D2均在100L发酵罐发酵后丰度增加。且烟酸、泛酸、抗坏血酸与维生素D2在100L发酵罐发酵中丰度相似。视黄醇(维生素A)与核黄素均在100L发酵罐发酵阶段丰度达到最高。
21种常见有机酸的代谢情况见图6。由图6可知，苹果酸、柠檬酸、草酰琥珀酸、水杨酸、衣康酸、2-异丙基苹果酸、氧己二酸、肌苷酸、D-乳酸、莽草酸、丁酸、鞣花酸和山梨酸的丰度在发酵后均高于未发酵组。
实施例3：Trichosporon asahii YD-1发酵上清滤液的稳定性测试
对实施例2制备的Trichosporon asahii YD-1发酵液进行菌体分离处理，得到发酵上清滤液；并对其时间稳定性、高温稳定性、酸碱稳定性和光照稳定性进行测定。
(1)将添加过苯氧乙醇-乙基己基甘油(化妆品用防腐剂)的Trichosporon asahiiYD-1发酵上清滤液分别在–20℃、4℃、常温以及45℃保藏三个月后，恢复常温观察，结果如图7所示。由图7所知，发酵上清滤液的颜色、气味均无异常，液体无分层现象，无沉淀，性质稳定。保藏三个月后，进行核黄素与有机酸含量的测定，结果如图8所示。由图8所知，核黄素与有机酸含量均无明显损失。
(2)将Trichosporon asahii YD-1发酵上清滤液放置在70℃的恒温震荡器中24h，测定发酵上清滤液中核黄素与有机酸的含量，结果如图9所示。由图9所知，上清滤液中核黄素与有机酸的含量均无明显变化。
(3)将Trichosporon asahii YD-1发酵上清滤液分别调整pH为3.0、4.0、5.0、6.0、7.0、8.0，随后测定发酵上清滤液中核黄素与有机酸的含量变化，结果如图10所示。由图10所知，随着pH的逐渐升高，发酵上清滤液中的核黄素与有机酸含量逐渐降低。这说明，Trichosporon asahii YD-1发酵上清滤液无法在pH高于4.00的环境下保藏，保藏时需控制体系pH值。
(4)将Trichosporon asahii YD-1发酵上清滤液在自然灯光条件下照射24h，测定发酵上清滤液中核黄素与有机酸的含量变化，结果如图11所示。由图11所知，24h内核黄素含量会快速下降，而有机酸含量基本不受影响。这是因为，光照对核黄素的苯环结构没有分解作用，但是苯环之外的环内共轭结构会在光照后发生破坏；且异咯嗪环见光极易分解，形成光黄素。故为了保证发酵液中的核黄素保持稳定，保存时需要保全程避光。
综上所述，Trichosporon asahii YD-1发酵上清滤在避光、pH≤4.00且温度不高于45℃的条件下稳定性佳，可以长期保藏。
实施例4：Trichosporon asahii YD-1发酵上清滤液的安全性测试
(1)发酵上清滤液对小鼠成纤维细胞(L929细胞)增殖的影响
将实施例3制备的发酵上清滤液用添加过10％(v/v)胎牛血清的RPMI-1640培养基分别稀释成10％、20％、40％、60％、80％、100％(v/v)的浓度，并用0.22μm的无菌针头滤器过滤后备用。将小鼠成纤维细胞(L929细胞)培养至贴壁状态后，弃去孔中上清液，添加含有上述不同浓度发酵液的培养液，空白对照组用相同体积的未添加胎牛血清的RPMI-1640培养基替代，每孔100μL，空白孔中无细胞。于37℃、5％CO2培养箱中培养48h。待样品与细胞作用时间达到48h，在96孔板中每孔加入10μL的5g/L MTT溶液，作用4h后弃上清液，再于每孔加入100μL DMSO溶液，在酶标仪中振荡15min，检测各孔吸光度值，波长为490nm。此实验需做3次平行，按下式计算细胞增殖抑制率。
细胞增殖抑制率＝(1-实验组平均吸光度值/对照组平均吸光度值)×100％
结果如图12所示，当Trichosporon asahii YD-1发酵上清滤液浓度为40％(v/v)时，L929细胞活力为89.92±5.09％，而当发酵上清滤液浓度为50％(v/v)时，L929细胞活力为69.37±2.99％。一般认为，当细胞活力高于80％，即证明供试药物对于细胞基本无毒性。故需要细化浓度梯度范围，进一步确定可以应用于化妆品的最佳Trichosporon asahiiYD-1发酵上清滤液浓度。
(2)发酵上清滤液对小鼠黑色素瘤细胞(B16F10细胞)增殖的影响
将实施例3制备的发酵上清滤液用添加过10％(v/v)胎牛血清的DMEM低碳酸氢钠培养基分别稀释成适宜浓度，并用0.22μm的无菌针头滤器过滤后备用。将小鼠黑色素瘤细胞(B16F10细胞)培养至贴壁状态后，弃去孔中上清液，添加含有上述不同浓度发酵上清滤液的培养液，空白对照组用相同体积的未添加胎牛血清的DMEM低碳酸氢钠培养基替代，每孔100μL，空白孔中无细胞。于37℃、5％CO2培养箱中培养48h。在96孔板中每孔加入10μL的5g/L MTT溶液，作用4h后弃上清液，再于每孔加入100μL DMSO溶液，在酶标仪中振荡15min，检测各孔吸光度值，波长为490nm。此实验需做3次平行。按下式计算细胞增殖抑制率。
结果如图13所示，当Trichosporon asahii YD-1发酵上清滤液浓度为42％(v/v)时，B16F10细胞活力为86.27±3.49％，而当发酵上清滤液浓度为49％(v/v)时，L929细胞活力为69.17±3.17％。
综合本实施例(1)(2)的结果可知，故从细胞层面来看，Trichosporon asahii YD-1发酵上清滤液使用浓度应当不高于42％(v/v)，使用该浓度可以充分保证其安全性。
(3)发酵液急性毒性评价
将28只小鼠分成7组，1组为健康组，其他6组分别配置20％、40％、100％、200％、500％和1000％(v/v)的100L发酵罐发酵48h的Trichosporon asahii YD-1上清滤液，均使用去离子水配制，健康组使用去离子水灌胃，灌胃给药量按小鼠最大给药体积：均为0.4ml/10g，连续14d观察小鼠的精神状态、食欲、大小便及分泌物形态、呼吸状态、皮肤被毛、不良反应及死亡情况，并记录每日小鼠体重，两周后，剖杀小鼠，观察出血、体腔积液和组织器官的病变情况。
结果如图14所示，将不同浓度的样品经口灌胃给药小鼠后，14天内只有空白组小鼠的体重基本呈逐步上升的趋势。而灌胃发酵上清液的各组小鼠体重均出现不同程度的体重波动，甚至在第14天，灌胃1000％(v/v)发酵上清液的小鼠平均体重低于灌胃当日体重。这说明，发酵上清液对于小鼠仍有一定影响。然而，其余组小鼠体重均在波动中逐渐上升，且通过观察小鼠的精神状态、食欲、大小便及分泌物形态、呼吸状态、皮肤被毛、不良反应及死亡情况，只有最高浓度组在灌胃首日，出现短暂安静并蜷缩身体的情况，且在第二日恢复正常状态。其余各组小鼠每日状态正常，且体重于14天内波动式增加，未观察到不良反应及死亡情况。试验结束后，剖检小鼠并未见脏器出现肉眼可见病变。在最大给药浓度为100％(v/v)时，小鼠未出现死亡；且无法测出Trichosporon asahii YD-1发酵上清滤液的LD50，这说明，其基本无毒副作用。根据急性毒性试验判定标准，Trichosporon asahii YD-1发酵上清滤液为无毒物质。
实施例5：Trichosporon asahii YD-1发酵上清滤液细胞层面美白功效测试
(1)发酵上清滤液对小鼠黑色素瘤细胞(B16F10细胞)黑色素含量的影响
将活化好的B16F10细胞以1×105个/mL的密度接种于TC处理过的24孔板中，每孔1mL，培养24h后，弃上清液。根据实施例4中结果，分别添加7％、14％、21％、28％、35％、42％(v/v)的发酵液和100μg/mL的曲酸溶液1mL；另外，再分别添加同样浓度梯度使用0.22μm针头滤器过滤过的发酵培养基溶液1mL，空白对照组添加1mL不含有FBS的DMEM低碳酸氢钠培养基代替样品溶液。设置培养温度为37℃，于5％CO2培养箱中培养24h后，弃上清液。每孔加入1mL胰酶，消化细胞5min后吹打成悬液。悬液离心弃上清液，沉淀中加入200μL含10％(v/v)二甲基亚砜的1mol/L NaOH溶液，80℃水浴中30min，直至细胞团彻底裂解和黑色素颗粒完全溶解，在酶标仪490nm处测吸光度值。按下式计算黑色素相对含量。
结果如图15所示，经历24h发酵液与未发酵培养基的处理，当未发酵培养基的浓度不高于21％(v/v)时，B16F10细胞中的黑色素相对含量在90％以上，几乎不影响细胞内的黑色素。而当未发酵培养基浓度达到42％(v/v)时，细胞中黑色素相对含量仍有58.19％。与之相比，发酵液对细胞中黑色素含量的影响要更加明显。发酵液浓度为7％(v/v)时，细胞中黑色素相对含量已经低于80％，而当发酵液浓度达到42％(v/v)时，细胞中黑色素相对含量为42.05％，甚至低于阳性对照的结果即100μg/mL的曲酸(其处理过的细胞，黑色素相对含量为50.78％)。这说明，发酵液相对于未发酵的培养基，对B16F10细胞的黑色素有明显的抑制作用。
(2)发酵上清滤液对小鼠黑色素瘤细胞(B16F10细胞)酪氨酸酶活性影响
将活化好的B16F10细胞以5×104个/mL的密度接种于TC处理过的96孔板中，每孔200μL，24h后弃上清液，分别添加7％、14％、21％、28％、35％、42％(v/v)的发酵液和100μg/mL的曲酸溶液200μL；另外分别添加同样浓度梯度使用0.22μm针头滤器过滤过的发酵培养基溶液200μL，空白对照组用不含有FBS的DMEM低碳酸氢钠培养基200μL代替样品溶液。设置培养温度37℃，于5％CO2培养箱中培养24h后，弃上清液。用pH 6.8的PBS溶液冲洗2次，每孔添加50μL的1％(v/v)TritonX-100溶液，放置在-80℃中40min，自然解冻后，在超声中破碎5min，每孔添加100μL的0.1％(w/v)L-DOPA溶液，在37℃下孵育2h，在酶标仪490nm处测吸光度值。按下式计算酪氨酸酶抑制率。
结果如图16所示，发酵液的酪氨酸酶抑制率要远高于未发酵培养基的酪氨酸酶抑制率，且当发酵液浓度高于35％(v/v)时，其酪氨酸酶抑制率接近80％，已经超过了100μg/mL的曲酸溶液。这说明，Trichosporon asahii YD-1发酵液具备优异的美白活性。
综上可知，采用本申请所述的海洋阿萨希丝孢酵母YD-1制备的发酵液，可以发酵得到富含核黄素和有机酸的发酵液，填补了现有技术的空白。此外，发明人研究发现，所述发酵液经菌体分离后得到的发酵上清滤液不但具备良好的稳定性和确定的安全性，而且其酪氨酸酶抑制率接近80％，具备优异的美白活性，在美白化妆品中应用前景广阔。同时，所述发酵液以葡萄糖和玉米浆为底物进行发酵生产，生产成本低且发酵工艺简单易操作，有利于产业化生产，市场价值不可估量。
Claims (8)
1.一株产核黄素和有机酸的海洋阿萨希丝孢酵母菌株，其特征在于：所述阿萨希丝孢酵母菌菌株(Trichosporon asahii)命名为YD-1，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M20231042，保藏日期为2023年6月19日。
2.如权利要求1所述的产核黄素和有机酸的海洋阿萨希丝孢酵母菌株的活化方法，其特征在于：将权利要求1所述的菌株YD-1接种到YPD固体培养基中，在28-30℃温度条件下培养1-2天。
3.一种包含权利要求1所述的海洋阿萨希丝孢酵母菌株YD-1的菌剂。
4.如权利要求1所述的海洋阿萨希丝孢酵母菌株的应用，其特征在于：将所述的阿萨希丝孢酵母菌株YD-1用于发酵生产富含核黄素和有机酸的发酵液。
5.根据权利要求4所述的海洋阿萨希丝孢酵母菌株的应用，其特征在于：具体步骤为：将权利要求1所述的阿萨希丝孢酵母菌株YD-1接入种子培养基中进行种子培养，将培养至对数生长期的种子液加入到发酵培养基，发酵完成后，分离菌体，得到富含核黄素和有机酸的发酵液。
6.根据权利要求5所述的阿萨希丝孢酵母菌菌株的应用，其特征在于：所述的种子培养基为葡萄糖20g/L，蛋白胨20g/L，酵母提取物10g/L；所述的发酵培养基为：葡萄糖35g/L，玉米浆20g/L。
7.根据权利要求6所述的阿萨希丝孢酵母菌菌株的应用，其特征在于：所述种子培养的温条件为28℃，培养时间为12-16h；所述的发酵条件为：发酵起始pH为3.20，发酵温度为28℃，搅拌速度为200rpm，通气量为1.5m3/h。
8.如权利要求4-7中任意一项制备得到的阿萨希丝孢酵母发酵液。
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