CN114480177B - 一株高产Levan果聚糖的产左聚糖微杆菌及Levan果聚糖的应用 - Google Patents
一株高产Levan果聚糖的产左聚糖微杆菌及Levan果聚糖的应用 Download PDFInfo
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Abstract
本发明公开了一株高产Levan果聚糖的产左聚糖微杆菌及Levan果聚糖的应用。所述菌株分类命名为产左聚糖微杆菌(Microbacteriumlaevaniformans)CN‑01,于2020年12月11日保藏至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.21356。Levan果聚糖由产左聚糖微杆菌(M.laevaniformans)CN‑01发酵而得,经发酵条件优化后,Levan果聚糖的产量达到35.2 g/L。实验证明,所述的由CN‑01分泌的Levan果聚糖具有良好的减脂功能,在减肥食品领域由良好的应用前景。
Description
技术领域
本发明涉及生物工程技术领域,特别涉及一种高产Levan果聚糖的产左聚糖微杆菌及Levan果聚糖的应用。
背景技术
Levan果聚糖是一类典型的果糖聚合物。不同来源及不同分子量的Levan果聚糖具备丰富的生物学功能。比如来源于M. laevaniformans的Levan(Mw=710 kDa)和来源于Zymomonasmobilis的Levan(Mw=570 kDa)对肉瘤180细胞有较强的抑制作用,而Gluconoacetobacterxylinus产生的Levan(Mw=40 kDa)的抑制效果却显著降低。低分子量的Levan果聚糖则可抑制腐败菌和致命菌的存活率。此外,研究人员从不同蜂蜜中分离得到多株产Levan的Bacillus subtilis,其中来自菌株K、M、E的Levan可抑制致病性禽流感病毒HPAI和H5N1的活性,而菌株A、M的Levan对40型腺病毒有抑制活性。
除了微生物来源的Levan,它还存在于植物中。植物来源的levan主要存在于草本植物,但是其天然含量低,提取和分离成本高,不适于产业化生产。微生物发酵液提取和酶法合成是目前大量获得Levan果聚糖的两种方法,其中微生物发酵液提取的Levan果聚糖产量和蔗糖转化率一般较低,且发酵液中同时存在的其他高聚物不利于Levan的规模化纯化;而酶法制备虽然产量高,但由于是体外反应,存在天然缺陷,即分子量低。比如2016年江南大学的硕士论文《Bacillus amyloliquefaciensH47果聚糖蔗糖酶基因的克隆、表达及其应用研究》中,酶法制备的Levan果聚糖分子量仅为 7.5 kDa,因此,筛选一株高产Levan果聚糖的菌株对于其商业化应用至关重要。
发明内容
本发明的目的是拓展高产Levan果聚糖的微生物资源,同时提供一种Levan果聚糖的应用。
为实现上述目的,现提供以下技术方案:
一株高产Levan果聚糖的产左聚糖微杆菌,分类命名为产左聚糖微杆菌(M.laevaniformans)CN-01,于2020年12月11日保藏至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. 21356。
所述菌株M. laevaniformans CN-01筛选于江苏省南京市浦口区珠江镇的水稻田中的水稻根际土壤,使用LB培养基分离得到。
在LB固体培养基上37 ℃培养16-24h观察发现菌落呈浅黄色、光滑、不透明、表面相对平整且边缘规整。
本发明Levan果聚糖的发酵方法按以下步骤进行:
挑取CN-01的单菌落至LB培养基(NaCl 10 g/L,酵母粉 5 g/L,蛋白胨 10 g/L)中,37 ℃,200 rpm,过夜培养;
将CN-01的种子液以4%(v/v)的转接量转接至发酵培养基(蔗糖 60 g/L,丙氨酸 4g/L,Na2HPO46.9 g/L,KH2PO43.0 g/L,NaCl 0.5 g/L,NH4Cl 1.0 g/L,MgSO4·7H2O 0.5 g/L,CaCl20.1 g/L)中,于28 ℃,200 rpm,培养48 h,发酵期间,用氨水将发酵液pH控制在7左右,发酵结束后,即得含Levan果聚糖的发酵液。
本发明Levan果聚糖的提取方法按以下步骤进行:
将上述含Levan果聚糖的发酵液用纯水稀释1倍体积后,除去菌体,获得上清液;
将上清液通过60 ℃减压蒸馏,浓缩至原始体积的1/3-1/2;
添加浓缩液2倍体积的95%的工业酒精,4 ℃过夜醇沉多糖;
将多糖复溶于纯水后按上述步骤再次醇沉,随后4000 ×g离心10 min,保留沉淀,干燥后即为Levan果聚糖。
本发明菌株制备得到的Levan果聚糖分子量为110 kDa。
按上述方法制备的Levan果聚糖在微生物制备Levan果聚糖中具有较高的C源利用率,在本发明一个实施例中,C源利用率达58.7%,Levan果聚糖的产量为35.2 g/L。
碳源利用率=多糖产量/培养基中的碳源添加量。
所述的Levan果聚糖具有降脂减肥的作用。
所述的Levan果聚糖在制备减脂药品中的应用。
有益效果:本发明筛选得到一株高产Levan果聚糖的产左聚糖微杆菌,该菌株具有高碳源利用率,发酵所获得的Levan果聚糖具有降脂减肥的作用,在制备降脂减肥的药品中具有广阔的应用前景。
附图说明
图1 菌株CN-01的菌落形态及进化树;
图2 不同碳源、氮源、pH和温度对Levan果聚糖产量的影响;
图3 碳源、氮源的不同浓度对Levan果聚糖产量的影响;
图4 Levan果聚糖的单糖组分色谱图;
图5 Levan果聚糖对高脂饲养小鼠的减脂效果。
实施方式
为使本发明的目的、特征、优点能够更加的明显和易懂,下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,下面所描述的实施例仅仅是本发明一部分实施例,而非全部实施例。基于本发明中的实施例,本领域的技术人员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1M. laevaniformansCN-01的筛选和鉴定
水稻根际土壤采集自江苏省南京市浦口区珠江镇的水稻田,将采取的水稻根系抖落松散的土块后,将根系剪下,置于50 mL 无菌水中。充分震荡后,取100μL涂布至含有40g/L蔗糖的LB固体培养基(NaCl 10 g/L,酵母粉 5 g/L,蛋白胨 10 g/L)中,于30 ℃,培养24 h。
挑取平板中菌落直径大,分泌粘液多的菌株,在LB固体平板中划线纯化,培养条件为:37 ℃,过夜培养。连续划线传代4次后,获得纯培养菌株。所述菌株在LB固体培养基上37℃培养16-24h观察发现菌落呈浅黄色、光滑、不透明、表面相对平整且边缘规整(附图1)。随后,挑取单菌落至液体LB培养基中,过夜培养后使用诺维赞基因组提取试剂盒FastPureBlood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (货号:DC112-01)提取CN-01的基因组,并使用27 F(5’-AGAGTTTGATCCTGGCTCAG-3’)和1492 R(5’-TACGGCTACCTTGTTACGACTT-3’)引物克隆CN-01的16S rDNA片段,送至通用生物(安徽)股份有限公司进行测序。CN-01的16S rDNA如SEQ ID NO.1所示。测序结果在NCBI网站上(https://blast.ncbi.nlm.nih.gov)经过BLASTn工具序列比对及通过MEGA7进化树分析后,确认其为M. laevaniformans(附图1)。
实施例2M. laevaniformansCN-01的发酵产Levan果聚糖的条件优化
CN-01的初始发酵培养基为葡萄糖40 g/L,Na2HPO46. 9 g/L,KH2PO43.0 g/L,NaCl0.5 g/L,NH4Cl 1.0 g/L,MgSO4·7H2O 0.5 g/L,CaCl20.1 g/L。首先考察不同碳源(葡萄糖,蔗糖,果糖,甘露糖,木糖,甘油,均替代初始发酵培养基中的葡萄糖),不同氮源(硫酸铵,蛋白胨,酵母粉,鱼粉蛋白胨,谷氨酸,丙氨酸,在最优C源的基础上均额外添加,终浓度为5 g/L),不同温度(20,25,26,28,30,35℃),不同pH(4,5,6,7,8,9)对CN-01发酵的影响。
实验结果如附图2所示,最佳碳源为蔗糖,最佳氮源为丙氨酸,最适温度为28℃,最适pH为7。
随后,再次通过单因素优化,进一步考察了不同蔗糖浓度(10,20,40,60,80,100g/L)和不同丙氨酸浓度(1,2,4,6,8,10 g/L)对Levan果聚糖产量的影响。
结果如附图3所示,最适蔗糖浓度为60 g/L,最适丙氨酸浓度为4 g/L,在此条件下,Levan果聚糖产量为35.2 g/L,碳源利用率58.7%。
实施例3 最佳条件下M. laevaniformansCN-01的发酵产Levan果聚糖
挑取CN-01的单菌落至LB培养基(NaCl 10 g/L,酵母粉 5 g/L,蛋白胨 10 g/L)中,37 ℃,200 rpm,过夜培养;
将CN-01的种子液以4%(v/v)的转接量转接至发酵培养基(蔗糖 60 g/L,丙氨酸 4g/L,Na2HPO46.9 g/L,KH2PO43.0 g/L,NaCl 0.5 g/L,NH4Cl 1.0 g/L,MgSO4·7H2O 0.5 g/L,CaCl20.1 g/L)中,于28 ℃,200 rpm,培养48 h,发酵期间,用氨水将发酵液pH控制在7左右,发酵结束后,即得含Levan果聚糖的发酵液。
发酵液中Levan果聚糖的提取方法按以下步骤进行:
将上述含Levan果聚糖的发酵液(1 L)用纯水稀释1倍体积后,通过板框压滤机除去菌体,获得上清液;将上清液通过60 ℃减压蒸馏,浓缩至400 mL;添加800 mL的95%的工业酒精,4 ℃过夜醇沉多糖。将多糖复溶于400 mL纯水后再次使用800 mL的95%的工业酒精醇沉,随后4000 ×g离心10 min,保留沉淀,干燥后即为Levan果聚糖。
实施例4 Levan果聚糖分子量及单糖组分鉴定
Levan果聚糖分子量通过GPC法测定。具体为:将Levan果聚糖样品(10 mg)溶解在1mL纯水中,随后用0.22-μm水系膜过滤。通过HPLC系统进行分析,所用色谱柱为Ultrahydrogel™ 500(7.8 × 300 mm)、Ultrahydrogel™(7.8 × 300 mm),检测器为蒸发光检测器,流动相为超纯水,流速为1 mL/min,柱温为35 ℃。使用不同分子量的葡聚糖作为标品,估算Levan果聚糖的分子量。
经测定,Levan果聚糖为110 kDa。
Levan果聚糖单糖组成通过HPLC法测定。
将Levan果聚糖样品(10 mg)溶解在1 mL纯水中,缓慢加入浓硫酸,使其终浓度为0.5 M,随后在80℃水解1 h。往水解液中加入过量碳酸钡,至无气泡生成。离心后取上清,即为胞外多糖的单糖水解液。水解液用0.22-μm水系膜过滤后,通过HPLC系统进行分析。所用色谱柱为Sugar-pak1,检测器为蒸发光检测器,流动相为超纯水,流速为0.5 mL/min,柱温为70 ℃。以葡萄糖,果糖和半乳糖为标准品,判断Levan果聚糖的单糖组成。
经测定,Levan果聚糖的由果糖组成(附图4),其中,A为标准品的液相色谱图,B为Levan果聚糖的单糖组成的液相色谱图。
实施例5 Levan果聚糖的减肥用途
将50只雄性C57BL/6小鼠(4周龄,体重为20 g左右)置于动物房内饲养,饲养温度为23 ± 3℃,湿度为50 ± 5%,光照周期为白天/黑夜:12h/12h。用普通饲料适应性喂养一周后(体重为23 g左右),将其随即分成5组,每组十只。具体分组如下:
T1组:饲喂普通饲料;
T2组,饲喂高脂饲料(含45%脂肪);
T3组,饲喂高脂饲料(含45%脂肪)+0.1% Levan果聚糖;
T4组,饲喂高脂饲料(含45%脂肪)+0.5% Levan果聚糖;
T5组,饲喂高脂饲料(含45%脂肪)+1% Levan果聚糖;
其中,Levan果聚糖按实施例2和3所述的方式发酵与制备所得。
试验期间,小鼠正常进食和饮水,持续饲喂12周。
实验结束后,记录小鼠质量,眼眶取血后,将小鼠颈椎脱臼处死。血液样品4 ℃离心(1500 rpm,15min),采用东芝全自动生化分析仪分析血清中的甘油三酯含量和总胆固醇含量。
实验结果如附图5所示,与T2组相比,T3、T4、T5处理组显著降低了高脂饲料饲喂条件下的体重,总胆固醇含量和甘油三酯含量。这说明,由M. laevaniformansCN-01发酵所得的Levan果聚糖具有良好的减脂作用。
序列表
<110> 苏州科宁多元醇有限公司
<120> 一株高产Levan果聚糖的产左聚糖微杆菌及其在产Levan果聚糖中的应用
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gttgctgatc tgcgattact agcgactccg acttcatgag gtcgagttgc agacctcaat 180
ccgaactggg accggctttt tgggattcgc tccacctcac ggtattgcag ccctttgtac 240
cggccattgt agcatgcgtg aagcccaaga cataaggggc atgatgattt gacgtcatcc 300
ccaccttcct ccgagttgac cccggcagta tcccatgagt tcccaccatt acgtgctggc 360
aacatagaac gagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct 420
gacgacaacc atgcaccacc tgtttacgag tgtccaaaga gttctacatt tctgcagcgt 480
tctcgtatat gtcaagcctt ggtaaggttc ttcgcgttgc atcgaattaa tccgcatgct 540
ccgccgcttg tgcgggtccc cgtcaattcc tttgagtttt agccttgcgg ccgtactccc 600
caggcgggga acttaatgcg ttagctgcgt cacggaatcc gtggaatgga ccccacaact 660
agttcccaac gtttacgggg tggactacca gggtatctaa gcctgtttgc tccccaccct 720
ttcgctcctc agcgtcagtt acggcccaga gatctgcctt cgccatcggt gttcctcctg 780
atatctgcgc attccaccgc tacaccagga attccaatct cccctaccgc actctagtct 840
gcccgtaccc actgcagacc cgaagttgag cctcgggatt tcacagcaga cgcgacaaac 900
cgcctacgag ctctttacgc ccaataattc cggataacgc ttgcgcccta cgtattaccg 960
cggctgctgg cacgtagtta gccggcgctt tttctgcagg taccgtcact ttcgcttctt 1020
ccctgctaaa agaggtttac aacccgaagg ccgtcatccc tcacgcggcg ttgctgcatc 1080
aggctttcgc ccattgtgca atattcccca ctgctgcctc ccgtaggagt ctgggccgtg 1140
tctcagtccc agtgtggccg gtcaccctct caggccggct acccgtcgac gccttggtga 1200
gccattacct caccaacaag ctgataggcc gcgagctcat ccctgaccaa aaaatctttc 1260
caaccgttga agatgcctcc acggttcgta tccagtatta gacgccgttt ccagcgctta 1320
tcccagagtc aggggcagat tgctcacgtg ttactcaccc gttcgccact gatccaccca 1380
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Claims (7)
1. 一株高产Levan果聚糖的产左聚糖微杆菌(Microbacteriumlaevaniformans)CN-01,其于2020年12月l1日保藏至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.21356。
2.权利要求1所述高产Levan果聚糖的产左聚糖微杆菌(Microbacteriumlaevaniformans)CN-01在产Levan果聚糖中的应用。
3. 根据权利要求2所述的应用,其特征在于,所述应用包含将产左聚糖微杆菌CN-01接种到发酵培养基中,于28 ℃,培养48 h,发酵结束后,即得含Levan果聚糖的发酵液。
4. 根据权利要求3所述的应用,其特征在于,所述发酵培养基由蔗糖 60 g/L,丙氨酸4 g/L,Na2HPO4 6.9 g/L,KH2PO4 3.0 g/L,NaCl 0.5 g/L,NH4Cl 1.0 g/L,MgSO4·7H2O 0.5g/L,CaCl2 0.1 g/L组成。
5.根据权利要求3所述的应用,其特征在于,所述应用进一步包含将含Levan果聚糖的发酵液除去菌体,上清液中加入乙醇,醇沉多糖,离心保留沉淀,干燥后即为Levan果聚糖。
6.权利要求1所述高产Levan果聚糖的产左聚糖微杆菌(Microbacteriumlaevaniformans)CN-01发酵生产得到的Levan果聚糖。
7.权利要求6所述的Levan果聚糖在制备减脂药品中的应用。
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