CN115093992A - 植物乳植杆菌GLP40及其在发酵转化制备稀有人参皂苷Rk3中的应用 - Google Patents
植物乳植杆菌GLP40及其在发酵转化制备稀有人参皂苷Rk3中的应用 Download PDFInfo
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- CN115093992A CN115093992A CN202210645474.6A CN202210645474A CN115093992A CN 115093992 A CN115093992 A CN 115093992A CN 202210645474 A CN202210645474 A CN 202210645474A CN 115093992 A CN115093992 A CN 115093992A
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- lactobacillus plantarum
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Abstract
植物乳植杆菌(Lactiplantibac illusplantarum)GLP40及其在发酵转化制备稀有人参皂苷Rk3中的应用，涉及益生菌发酵领域，该菌株已于2022年03月09日保藏于中国典型培养物保藏中心，保藏编号为：CCTCCNO：M2022222。本发明确定了菌株GLP40发酵转化制备稀有人参皂苷Rk3的新工艺，同时采用HPLC法对转化后的稀有人参皂苷Rk3进行了鉴定。该工艺能以人参总皂苷提取物为原料，获得含量较高的稀有人参皂苷Rk3，该工艺具有安全、高效、成本低廉、反应温和等优点，该工艺适合规模化工业生产，所制备的稀有人参皂苷Rk3用途广泛，应用前景广阔。
Description
技术领域
本发明涉及益生菌发酵技术领域，具体涉及一种植物乳植杆菌GLP40及其在发酵转化制备稀有人参皂苷Rk3中的应用。
背景技术
目前，人参皂苷向稀有人参皂苷的转化途径有很多，例如在酸性环境下加热酸水解，制备稀有人参皂苷。但是这种转化途径特异性低，反应剧烈，且酸液无法回收，造成大量的环境污染。也有学者使用微波辅助降解人参皂苷，使其向稀有人参皂苷转化，这种方式的缺点是高能源、高成本，不利于大规模工业生产。
近些年来，微生物转化天然产物的研究逐渐兴起，有学者使用细菌、真菌等微生物对人参皂苷进行发酵，均能得到C-K、Rg3等稀有人参皂苷，但其使用的发酵菌种来源不明，甚至是使用对人体有害的致病菌，这显著降低了产品的安全性。已有研究表明，使用能够产生β-葡萄糖苷酶和β-半乳糖苷酶的乳酸菌如干酪乳杆菌、副干酪乳杆菌、嗜酸乳杆菌等，均能够以人参总皂苷提取物为底物，对其进行发酵转化，并成功得到Rg3、F2、C-K等稀有人参皂苷，但利用这些菌种转化稀有人参皂苷Rk3的报道并不多。
发明内容
有鉴于此，本发明的目的在于提供一种植物乳植杆菌GLP40及其在发酵转化制备稀有人参皂苷Rk3中的应用，为稀有人参皂苷Rk3的制备提供一条新的途径。
为了实现上述发明目的，本发明提供了如下技术方案：
本发明的植物乳植杆菌(Lactiplantibacillus plantarum)GLP40，该菌株已于2022年03月09日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M2022222。
本发明的植物乳植杆菌(Lactiplantibacillus plantarum)GLP40在发酵转化制备稀有人参皂苷Rk3中的应用。
作为优选的实施方式，包括以下步骤：
制备植物乳植杆菌(Lactiplantibacillus plantarum)GLP40菌悬液，将其接种至灭菌后的液体发酵培养基，加入经过滤除菌的人参总皂苷提取物共同发酵培养；所得发酵产物依次经低温减压浓缩、冷冻干燥、萃取、离心后，所得上清再经减压回收溶剂、冷冻干燥后，获得含有稀有人参皂苷Rk3的干燥产物；采用HPLC法对干燥产物进行鉴定。
作为优选的实施方式，所述植物乳植杆菌(Lactiplantibacillus plantarum)GLP40菌悬液的制备方法如下：
将植物乳植杆菌(Lactiplantibacillus plantarum)GLP40接种于液体MRS培养基，37～45℃静置培养16～25h，4～10℃、4000～8000rpm离心5～10min，收集菌体沉淀；用发酵灭菌培养基水溶液悬浮，调整活菌数为1.0×109～1.0×1010CFU/ml，获得植物乳植杆菌(Lactiplantibacillus plantarum)GLP40菌悬液。
作为优选的实施方式，所述人参总皂苷提取物的制备方法如下：
将人参根粉经80～100℃热水提取，所得提取液经减压浓缩、冷冻干燥后，获得粗皂苷粗提物，再经D101大孔树脂柱层析，以不同浓度乙醇洗脱，收集洗脱液，回收溶剂后进行冷冻干燥获得人参总皂苷提取物。
作为优选的实施方式，所述液体发酵培养基包括：葡萄糖2～5g/L，酵母浸粉1～3g/L，pH6.0～7.0，121℃灭菌15min。
作为优选的实施方式，所述液体发酵培养基中，人参总皂苷提取物的添加量为10～20g/L，所述植物乳植杆菌(Lactiplantibacillus plantarum)GLP40菌悬液的接种量为10～30ml/L。
作为优选的实施方式，所述共同发酵培养的温度为37～42℃，静置发酵7～14天。
作为优选的实施方式，所述发酵产物依次经70℃低温减压浓缩，冷冻干燥，甲醇萃取，10000rpm、15min、4℃离心后获得上清。
作为优选的实施方式，所述采用HPLC法对干燥产物进行鉴定的具体过程如下：
将含有稀有人参皂苷Rk3的干燥产物溶于甲醇，经0.22μm微孔滤膜过滤后进行HPLC色谱分析，色谱柱为Agilent pursuit5 SB-C18色谱柱，进样量20μL，流速1mL/min，柱温30℃，检测波长203nm；流动相为A：水，B：乙腈，0～40min 18～21％(B)；40～42min 21～26％(B)；42～46min 26～32％(B)；46～66min 32～34％(B)；66～71min 34～38％(B)；71～77.70min 38.0～49.1％(B)；77.70～82min 49.1％(B)；82～83min 49.1～50.6％(B)；83～88min 50.6～59.6％(B)；88～89.80min 59.6～65.0％(B)；89.80～97min 65％(B)；97～102min 65～75％(B)；102～110min 75～85％(B)；110～115min 85％(B)；115～125min 85～18％(B)；125～130min 18.0％(B)。
本发明的有益效果是：
本发明筛选获得1株能将人参皂苷Re和Rg1生物转化为稀有人参皂苷Rk3的乳杆菌新菌株GLP40，同时对该菌株GLP40进行了鉴定，结果为植物乳植杆菌(Lactiplantibacillus plantarum)，并已于2022年03月09日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M2022222。
本发明确定了菌株GLP40发酵转化制备稀有人参皂苷Rk3的新工艺，同时采用HPLC法对转化后的稀有人参皂苷Rk3进行了鉴定。该工艺能够以人参总皂苷提取物为原料，获得含量较高的稀有人参皂苷Rk3，该工艺具有安全、高效、成本低廉、反应温和等优点，该工艺适合规模化工业生产，所制备的稀有人参皂苷Rk3用途广泛，应用前景广阔。
附图说明
图1为稀有人参皂苷Rk3的生物转化合成路线。
图2为人参皂苷Rk3标准品的HPLC色谱图。
图3为发酵前实施例2步骤(2)制备的人参总皂苷提取物的HPLC色谱图。
图4为发酵后实施例2步骤(3)制备的含有稀有人参皂苷Rk3的干燥产物的HPLC色谱图。
具体实施方式
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，所描述的实施例仅仅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做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、鉴定和保藏
1、菌株的分离
分离菌株所用样品及来源：2021年9月采集吉林省延吉市传统发酵的东北酸菜样品。将采集的样品经研磨、梯度稀释后，涂布于乳杆菌选择性固体培养基平板，于37℃培养48h，挑取平板上的典型菌落(乳白色、圆形、边缘整齐、凸起、不透明、表面湿润光滑)，划线培养MRS琼脂平板，分离得到纯菌落。纯培养的菌种接种到液体MRS培养基中培养，然后加入60％甘油，-80℃冰箱保存。将其中1株乳杆菌命名为GLP40。
2、菌株的鉴定
GLP40的生理生化鉴定结果：革兰氏染色阳性，过氧化氢酶试验阴性，联苯胺试验阴性，吲哚试验阴性，乙酰甲基甲醇试验阳性；不水解淀粉，不液化明胶，不产生硫化氢，发酵葡萄糖产酸不产气；不运动的杆菌；在15℃和45℃能够生长，最适生长温度37～42℃；适宜pH为5.0～7.0；耐受6.5％NaCl；GLP40在液体MRS培养基中呈均匀浑浊生长，久置菌体呈白色沉淀。
GLP40的API 50CH(法国梅里埃公司)鉴定结果如下表：
| 碳水化合物 | GLP40 | 碳水化合物 | GLP40 | 碳水化合物 | GLP40 | 碳水化合物 | GLP40 | 碳水化合物 | GLP40 |
| S-甘露醇 | - | D-葡萄糖 | + | 山梨醇 | - | D-麦芽糖 | - | 木糖醇 | - |
| 赤藻糖醇 | - | D-果糖 | + | 甲基-D-吡喃甘露糖苷 | - | D-乳糖 | + | D-龙胆二糖 | + |
| D-***糖 | - | D-甘露醇 | + | 甲基-D-吡喃葡萄糖苷 | - | D-蜜二糖 | + | D-土伦糖 | - |
| L-***糖 | - | L-山梨糖 | - | N-乙酰-葡糖胺 | + | D-蔗糖 | + | D-来苏糖 | - |
| D-核糖 | + | L-鼠李糖 | - | 苦杏仁甙 | + | D-海藻糖 | + | D-塔格糖 | - |
| D-木糖 | - | 卫茅醇 | - | ARBULIN | - | 菊糖 | - | D-岩藻糖 | - |
| L-木糖 | - | 肌醇 | - | 七叶灵柠檬酸铁铵 | - | D-松三糖 | - | L-岩藻糖 | - |
| D-侧金盏花醇Ⅰ | - | 甘露醇 | + | 2-酮基-葡萄糖酸盐 | - | D-棉子糖 | + | D-***糖 | + |
| 葡萄糖酸钾 | + | D-纤维二糖 | + | 5-酮基-葡萄糖酸盐 | - | 淀粉 | - | L-***糖 | - |
| D-半乳糖 | + | 水杨苷 | + | 甲基-βD–吡喃木糖苷 | - | 糖原 | - |
上表中，“+”表示菌株为阳性反应；“-”表示菌株为阴性反应。
16S rDNA和pheS基因序列扩增和比对。采用CTAB法提取基因组DNA，根据基因序列的保守区域分别设计16S rDNA引物(27F：5'-AGAGTTTGATCCTGGCTCAG-3'；1492R：5'-GGTTACCTTGTTACGACT T-3')和pheS引物(p-F：5'-CCGTGAAGAACTGGAACA-3'，p-R：5'-CCTAACCCAAAGGCAAAA-3')，进行PCR扩增和产物测序后，通过BLAST将获得的菌株16S rDNA序列和pheS序列与GenBank库中已知菌株的序列信息进行同源性比对分析，鉴定待测菌株；以16S rDNA序列和pheS序列同源性大于99％为标准进行属种归类。所获得的16s rDNA序列如序列表中的SEQ ID NO:1所示，所获得的pheS序列如序列表中的SEQ ID NO:2所示。将16srDNA序列和pheS序列通过BLAST程序在GenBank数据库中进行同源性比对分析，发现GLP40与植物乳植杆菌Lac tobacillus plantarum的同源性达到99％。
根据以上结果，菌株GLP40被鉴定为植物乳植杆菌(Lactiplantibacillusplantarum)。
3、菌株的保藏
本发明的植物乳植杆菌(Lactiplantibacillus plantarum)GLP40，已于2022年03月09日保藏于中国典型培养物保藏中心，简称CCTCC，地址为：湖北省武汉市武昌区八一路299号武汉大学校内(武汉大学保藏中心)，保藏编号为：CCTCC NO：M2022222。
实施例2利用植物乳植杆菌(Lactiplantibacillus plantarum)GLP40发酵转化制备稀有人参皂苷Rk3
1、植物乳植杆菌(Lactiplantibacillus plantarum)GLP40菌悬液的制备
植物乳植杆菌(Lactiplantibacillus plantarum)GLP40(以下均用GLP40表示)接种于液体MRS培养基，37℃静置培养16h，4℃、6000rpm离心8min，收集菌体沉淀；用发酵灭菌培养基水溶液悬浮，调整活菌数为1.0×109CFU/mL，获得GLP40菌悬液。
2、人参总皂苷提取物的制备
将人参根粉(购自西安神农生物科技有限公司)经热水(80℃)提取，所得提取液经减压浓缩、冷冻干燥后，获得粗皂苷粗提物，再经D101大孔树脂柱层析，以不同浓度乙醇(0～90％)洗脱，收集洗脱液，回收溶剂后进行冷冻干燥获得人参总皂苷提取物。
3、发酵转化
液体发酵培养基包括葡萄糖3g/L，酵母浸粉1g/L，pH6.6，121℃灭菌15min。液体发酵培养基中，人参总皂苷提取物的添加量为10g/L，并按10ml/L的接种量接种GLP40菌悬液，37℃静置发酵14天。发酵结束后，所得发酵产物低温(70℃)减压浓缩，冷冻干燥，产物用甲醇萃取，离心(10000rpm，15min，4℃)，上清减压回收溶剂后进行冷冻干燥，获得含有稀有人参皂苷Rk3的干燥产物，该干燥产物为含有稀有人参皂苷Rk3的混合物。
4、在利用植物乳植杆菌(Lactiplantibacillus plantarum)GLP40发酵转化制备稀有人参皂苷Rk3的过程中，稀有人参皂苷Rk3的生物转化合成路线如图1所示：人参皂苷Re在植物乳植杆菌(Lactiplantibacillus plantarum)GLP40产的β-D-葡葡萄糖苷酶作用下水解生成人参皂苷F4和人参皂苷Rg6，人参皂苷Rg6进一步在植物乳植杆菌(Lactiplantibacillus plantarum)GLP40产的α-L-鼠李糖苷酶作用下转化生成稀有人参皂苷Rk3；稀有人参皂苷Rk3的另一条转化路线是由人参皂苷Rg1在β-D-葡葡萄糖苷酶水解20位的葡萄糖后脱水生成。
实施例3稀有人参皂苷Rk3的鉴定
1、高效液相色谱法(HPLC)检测稀有人参皂苷Rk3
分别将实施例2步骤(2)制备的人参总皂苷提取物以及实施例2步骤(3)制备的含有稀有人参皂苷Rk3的干燥产物溶于甲醇，经0.22μm微孔滤膜过滤后用于HPLC色谱分析。
HPLC色谱分析方法：色谱柱为Agilent pursuit5 SB-C18色谱柱，进样量20μL，流速1mL/min，柱温30℃，检测波长203nm。流动相为A：水，B：乙腈，0～40min 18～21％(B)；40～42min 21～26％(B)；42～46min26～32％(B)；46～66min 32～34％(B)；66～71min 34～38％(B)；71～77.70min 38.0～49.1％(B)；77.70～82min 49.1％(B)；82～83min49.1～50.6％(B)；83～88min 50.6～59.6％(B)；88～89.80min 59.6～65.0％(B)；89.80～97min65％(B)；97～102min 65～75％(B)；102～110min 75～85％(B)；110～115min 85％(B)；115～125min 85～18％(B)；125～130min 18.0％(B)。
2、鉴定结果
如图2-图4所示，通过对发酵前后产物中人参皂苷类成分含量的变化分析可知，稀有人参皂苷Rk3在HPLC色谱条件下的保留时间与人参皂苷标准品(购自上海源叶生物技术有限公司，HPLC≥98％)的保留时间一致。由此证明，人参总皂苷提取物中的人参皂苷ReRe和Rg1可经过植物乳植杆菌(Lactiplantibacillus plantarum)GLP40发酵转化为稀有人参皂苷Rk3。
本发明公开了一种植物乳植杆菌GLP40及其在发酵转化制备稀有人参皂苷Rk3中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的产品已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的产品进行改动或适当变更与组合，来实现和应用本发明技术。
序列表
<110> 吉林省农业科学院
<120> 植物乳植杆菌GLP40及其在发酵转化制备稀有人参皂苷Rk3中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1415
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aggacgaacg ctggcggcgt gcctaataca tgcaagtcga acgaactctg gtattgattg 60
gtgcttgcat catgatttac atttgagtga gtggcgaact ggtgagtaac acgtgggaaa 120
cctgcccaga agcgggggat aacacctgga aacagatgct aataccgcat aacaacttgg 180
accgcatggt ccgagtttga aagatggctt cggctatcac ttttggatgg tcccgcggcg 240
tattagctag atggtgaggt aacggctcac catggcaatg atacgtagcc gacctgagag 300
ggtaatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg 360
gaatcttcca caatggacga aagtctgatg gagcaacgcc gcgtgagtga agaagggttt 420
cggctcgtaa aactctgttg ttaaagaaga acatatctga gagtaactgt tcaggtattg 480
acggtattta accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 540
tggcaagcgt tgtccggatt tattgggcgt aaagcgagcg caggcggttt tttaagtctg 600
atgtgaaagc cttcggctca accgaagaag tgcatcggaa actgggaaac ttgagtgcag 660
aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720
gtggcgaagg cggctgtctg gtctgtaact gacgctgagg ctcgaaagta tgggtagcaa 780
acaggattag ataccctggt agtccatacc gtaaacgatg aatgctaagt gttggagggt 840
ttccgccctt cagtgctgca gctaacgcat taagcattcc gcctggggag tacggccgca 900
aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 960
tcgaagctac gcgaagaacc ttaccaggtc ttgacatact atgcaaatct aagagattag 1020
acgttccctt cggggacatg gatacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080
gatgttgggt taagtcccgc aacgagcgca acccttatta tcagttgcca gcattaagtt 1140
gggcactctg gtgagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca 1200
tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa cgagttgcga 1260
actcgcgaga gtaagctaat ctcttaaagc cattctcagt tcggattgta ggctgcaact 1320
cgcctacatg aagtcggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt 1380
cccgggcctt gtacacaccg cccgtcacac catga 1415
<210> 2
<211> 702
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cgtgaagaaa tggaacaagc agccgttaat gagcaattgg ctgccgaaaa actggacgtg 60
acgttaccgg gtcgggaagt tccacaaggt cagcctcacg tgattaccca gattattact 120
gaattggaag atctatttat gggaatgggc tatcaaattg ttgatggtga tgaagttgaa 180
gaggattact acaactttga acggttgaac ttaccgaagg accatcccgc ccgtgacatg 240
caagacacgt tctatattac caaagacgtg ctactacgca cgcagacgtc tgctgatcag 300
ccgcggtcac ttgaaaatca cgatttttct aaaggaccgc tgaaggtctt gtcacctggc 360
cgcgtttatc ggcgtgatac ggatgatgca acccattccc atcaatttca tcaaattgaa 420
gggttagtcg tggacaagca tattacgatg gctgatttga agggcacctt aattctggtt 480
gccaagactt tgtttggcga tcaattcgat gttcggctac ggccaagctt ctttccattc 540
acggaaccat ccgtagaagc tgatgtaact tgctttaatt gcaatggcaa gggctgtgca 600
atctgtaagc aaacgggttg gatcgaagta ctgggtgccg gcatggttca cccccacgtg 660
ttagaaatgt ctggcattga tccagaagat tatggtggtt tt 702
Claims (10)
1.植物乳植杆菌(Lactiplantibacillus plantarum)GLP40，其特征在于，该菌株已于2022年03月09日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M2022222。
2.如权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)GLP40在发酵转化制备稀有人参皂苷Rk3中的应用。
3.根据权利要求2所述的应用，其特征在于，包括以下步骤：
制备植物乳植杆菌(Lactiplantibacillus plantarum)GLP40菌悬液，将其接种至灭菌后的液体发酵培养基，加入经过滤除菌的人参总皂苷提取物共同发酵培养；所得发酵产物依次经低温减压浓缩、冷冻干燥、萃取、离心后，所得上清再经减压回收溶剂、冷冻干燥后，获得含有稀有人参皂苷Rk3的干燥产物；采用HPLC法对干燥产物进行鉴定。
4.根据权利要求3所述的应用，其特征在于，所述植物乳植杆菌(Lactiplantibacillusplantarum)GLP40菌悬液的制备方法如下：
将植物乳植杆菌(Lactiplantibacillus plantarum)GLP40接种于液体MRS培养基，37～45℃静置培养16～25h，4～10℃、4000～8000rpm离心5～10min，收集菌体沉淀；用发酵灭菌培养基水溶液悬浮，调整活菌数为1.0×109～1.0×1010CFU/ml，获得植物乳植杆菌(Lactiplantibacillus plantarum)GLP40菌悬液。
5.根据权利要求3所述的应用，其特征在于，所述人参总皂苷提取物的制备方法如下：
将人参根粉经80～100℃热水提取，所得提取液经减压浓缩、冷冻干燥后，获得粗皂苷粗提物，再经D101大孔树脂柱层析，以不同浓度乙醇洗脱，收集洗脱液，回收溶剂后进行冷冻干燥获得人参总皂苷提取物。
6.根据权利要求3所述的应用，其特征在于，所述液体发酵培养基包括：葡萄糖2～5g/L，酵母浸粉1～3g/L，pH6.0～7.0，121℃灭菌15min。
7.根据权利要求3所述的应用，其特征在于，所述液体发酵培养基中，人参总皂苷提取物的添加量为10～20g/L，所述植物乳植杆菌(Lactiplantibacillus plantarum)GLP40菌悬液的接种量为10～30ml/L。
8.根据权利要求3所述的应用，其特征在于，所述共同发酵培养的温度为37～42℃，静置发酵7～14天。
9.根据权利要求3所述的应用，其特征在于，所述发酵产物依次经70℃低温减压浓缩，冷冻干燥，甲醇萃取，10000rpm、15min、4℃离心后获得上清。
10.根据权利要求3所述的应用，其特征在于，所述采用HPLC法对干燥产物进行鉴定的具体过程如下：
将含有稀有人参皂苷Rk3的干燥产物溶于甲醇，经0.22μm微孔滤膜过滤后进行HPLC色谱分析，色谱柱为Agilent pursuit5 SB-C18色谱柱，进样量20μL，流速1mL/min，柱温30℃，检测波长203nm；流动相为A：水，B：乙腈，0～40min 18～21％(B)；40～42min 21～26％(B)；42～46min 26～32％(B)；46～66min 32～34％(B)；66～71min 34～38％(B)；71～77.70min 38.0～49.1％(B)；77.70～82min 49.1％(B)；82～83min49.1～50.6％(B)；83～88min 50.6～59.6％(B)；88～89.80min 59.6～65.0％(B)；89.80～97min 65％(B)；97～102min 65～75％(B)；102～110min 75～85％(B)；110～115min 85％(B)；115～125min 85～18％(B)；125～130min 18.0％(B)。
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Non-Patent Citations (1)
| Title |
|---|
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Effective date of registration: 20240430 Address after: Warehouse 3 for the annual production of 300000 sets of automotive door panel systems and automotive pipe fittings connection systems at No. 8755 Xihu Road, Changchun Automotive Economic and Technological Development Zone, Jilin Province, 130000 Patentee after: Jilin Huayu Kangyuan Biotechnology Co.,Ltd. Country or region after: China Address before: 130000 no.1363 ecological street, Jingyue high tech Development Zone, Changchun, Jilin Province Patentee before: Jilin Academy of Agricultural Sciences (China Agricultural Science and Technology Northeast Innovation Center) Country or region before: China |
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