CN113430126A - 一株短梗霉及用它制备黑色素多糖的方法 - Google Patents
一株短梗霉及用它制备黑色素多糖的方法 Download PDFInfo
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- CN113430126A CN113430126A CN202110919309.0A CN202110919309A CN113430126A CN 113430126 A CN113430126 A CN 113430126A CN 202110919309 A CN202110919309 A CN 202110919309A CN 113430126 A CN113430126 A CN 113430126A
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- polysaccharide
- melanin
- fermentation
- strain
- liquid
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Abstract
本发明公开了一株短梗霉及用它制备黑色素多糖的方法。本发明从济南南郊菜园分离到一株高产黑色素多糖的短梗霉(Aureobasidium sp.)LHS‑m022,该菌株已于2021年7月15日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.23050。采用该菌株发酵法生产微生物黑色素多糖,所采用的发酵基质廉价易得、发酵工艺及条件科学合理,具有发酵周期短、产量高的优势,产物黑色素多糖具有耐高温、絮凝率高、抗氧化能力强、对光照稳定等优点。
Description
技术领域
本发明涉及一株短梗霉及用它制备黑色素多糖的方法,属于生物技术与生物工程领域。
背景技术
多糖是一种绿色、可再生的生物资源。在历史几千年的长河中,多糖为人类生存和发展提供了无限价值的产品。多糖是构成海洋甲壳动物、植物、海藻及微生物细胞壁的主要结构成分,储存碳源和能量。除此外,多糖还行使着其它多种生理功能,如可以保存和表达遗传信息、抵御其它细胞攻击和内外部有害环境的影响、传导生物及非生物信号。多糖具有获取方便、价格便宜、用途广泛的优势。
在过去的60年里,科学家将微生物作为一种生产多糖的自然资源。这是因为用微生物通过发酵工程的方式进行多糖生产,可以做到大规模、高速率、清洁化、自动化生产。微生物多糖是一种取之不尽的绿色资源,是一种能够可持续性发展的社会资源。微生物多糖由于其独特的物理和流变特性,被广泛应用在食品工业中作为增稠剂、稳定剂、凝胶剂和乳化剂。近年来,人们对微生物多糖的生物活性越来越感兴趣,如微生物多糖的抗病毒、免疫刺激、抗炎症活性,抗氧化作用和抗微生物作用。除此以外,深入研究与开发的应用新领域包括用作生物絮凝剂、生物吸收剂、重金属去除剂、药物释放剂等。
自然界的生物黑色素是天然黑色素,分布于动物、植物和微生物中,是一类复杂的酚类或吲哚类非均质的多聚物结构。近些年来,随着对生物资源的广泛和深入研究,生物黑色素的开发已经引起了研究者们广泛的关注,其特有的结构和功效已被逐步开发应用于食品、生物、化妆品、保健品、医学、环境、材料等领域。
科学研究发现,某些特殊的细胞既能产生黑色素又能产生多糖,在代谢上多糖的存在引导了黑色素的合成,黑色素的合成又促进了多糖产生,黑色素与多糖联合在一起形成了结构上的复合物。这种复合物结构形式从代谢方面促进了产物合成,从活性方面增强或增加了生物功能。研究发现,天然黑色素主要是吲哚物质,真菌构巢曲霉细胞壁中黑色素与氨基多糖结合的复合物能够抵抗微生物和外切β-D-1,3-葡聚糖酶的裂解(BULLAT.Journal of General Microbiology.1970,63,75-94)。从大米和米曲霉发酵制成的黑醋获取的黑色素葡聚糖复合物除具有抗氧化、防结肠炎发展、调整认知紊乱外,还具有抗肥胖的潜力,能够显著抑制前脂肪细胞的脂肪形成(SUZUKI E.et al.Journal ofFunctional Foods.2020,72,1-7.)。微生物产生的黑色素多糖复合物是特殊微生物的特殊生理现象,这种黑色素与多糖结构物质的复合势必会带来生理功能的拓宽和加强。对黑色素多糖的研究与产品开发潜在光明的前景和巨大的经济价值。
发明内容
针对上述问题,本发明提供了一株短梗霉及用它制备黑色素多糖的方法。本发明从济南南郊菜园分离到一株短梗霉(Aureobasidium sp.)LHS-m022,该菌株合成黑色素多糖的能力强、发酵周期短,产量高,得到的黑色素多糖耐高温,絮凝活性高,抗氧化性强,面团着色效果好。
本发明所提供的菌株为短梗霉(Aureobasidium sp.)LHS-m022,它是从山东省济南市南郊的菜园土壤中分离得到的,该菌株已于2021年7月15日保藏于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),保藏编号为CGMCC No.23050。该菌株可以制备黑色素多糖。
本发明还公开了采用短梗霉菌株LHS-m022制备黑色素多糖的方法,其特征是,
(1)液体菌种培养
将活化培养的斜面菌种接种于液体菌种培养基中,在24-30℃、150-180r/min条件下震荡培养18-28h,得液体菌种备用;
液体菌种培养基的组分及用量为(g/L):葡萄糖15-25,玉米浆1-3,K2HPO4 2-5,KH2PO41-2.5,MgSO4 0.5-1.5,FeSO4 0.0005-0.003,pH6.0-7.5。
(2)发酵培养
将液体菌种按6-10%(体积比)的接种量接种于发酵培养基,在挡板三角瓶中进行发酵培养66-72h,发酵结束得发酵液,将发酵液过滤去除菌体,得透明液体黑色素多糖。
发酵控制条件为:500mL挡板三角瓶分装培养基的量为100mL、培养温度为26-30℃、摇瓶机旋转转速为150-220r/min。
发酵培养基的组分及用量为(g/L):葡萄糖50-65,玉米浆2.5-4.5,K2HPO4 4-7,KH2PO4 1-3,MgSO4 0.8-1.5,MnCl2 0.004-0.009,FeSO4 0.0005-0.003,CuSO4 0.01-0.05,L-酪氨酸0.5-1.5,pH 6.0-7.5。
该方法制备的黑色素多糖为黑色素与葡萄糖、甘露糖、半乳糖、核糖、葡萄糖醛酸、半乳糖醛酸复合形成的生物杂多糖。其可溶于水,不溶于乙醇、丁醇、乙醚、丙酮。其粘度≥200mPa·S,OD300nm≥8,黑色素多糖含量≥3.0%。
该微生物黑色素多糖的用途及使用方法为:
(1)可用作生物絮凝剂,以液体状态直接搅拌混入地表水、生活污水、工业污水、生态湖水、养殖废水、发酵培养液等中进行絮凝;
(2)直接作为食品黑色素(如馒头、面条等)的着色剂;
(3)作为原料,制备补血用保健品-有机黑色素多糖铁,制备补钙用保健品-有机黑色素多糖钙,生产抗氧化保健品等。
(4)生产防晒(黑色素多糖能吸收可见光和紫外线,光稳定性特别强!)和/或抗氧化化妆品。
本发明的技术效果是:本发明筛选到了一株高产微生物黑色素多糖的短梗霉LHS-m022,采用该菌株发酵法生产微生物黑色素多糖,所采用的发酵基质廉价易得、发酵工艺及条件科学合理,具有发酵周期短(66-72h)、产量高(≥3%)的优势,产物黑色素多糖具有耐高温(121℃25min)、絮凝率高(≥94%)、抗氧化能力强(可达99%)、对光照稳定等优点,因此具有广阔的开发前景和很好的应用价值。
附图说明
图1为菌株LHS-m022平板单菌落图;
图2为菌株LHS-m022在光学学显微镜下的菌丝图;
图3为菌株LHS-m022的摇瓶发酵液;
图4为菌株LHS-m022的发酵产物(液体黑色素多糖);
图5为本发明黑色素多糖的FTIR(红外光谱)图;
图6为本发明黑色素多糖的HPLC(高效液相)多糖组分图谱;
图7为加入黑色素多糖蒸的小麦面馒头。
具体实施方式
以下结合实施例和附图对本发明进行详细说明。
黑色素多糖产量的测定方法为:将液体黑色素多糖溶液,在搅拌条件下用1.3倍无水乙醇进行沉淀,以5000r/min离心5min获得乙醇沉淀物,然后用6倍无水乙醇充分洗涤乙醇沉淀物,以5000r/min离心5min获得醇洗沉淀物,然后在40℃真空干燥6-12h,称干重。
絮凝率的测定方法为:配制(重量比):高岭土为0.4%、CaCl2为0.1%、pH7的高岭土(奉城牌,上海奉贤奉城试剂厂)悬液,加入混凝试验搅拌器的反应杯中,混匀,备用。测定时每100mL悬液中加入40μL样品,以120r/min的搅拌转速快速混合40s,再以60r/min的搅拌转速缓慢混合120s。然后,静止5min。吸取上清液,测定波长550nm下的OD值B。加蒸馏水替代样品重复以上操作测得OD值A。絮凝率(FR)={(A-B)/A}×100%。
抗氧化活性的测定方法:采用DPPH(2,2-二苯基-2-苦基肼基)法。在15mm×150mm试管中加入水2mL、2×10-4mol/L DPPH乙醇溶液2mL,混匀,室温下反应30min,测定波长517nm下的OD值A。样品2mL+2×10-4mol/L DPPH乙醇溶液2mL反应测得OD值B。样品2mL+水2mL,混匀,测得OD值C。抗氧化活性=[A-(B-C)]/A×100%。
实施例1:菌株的筛选
(1)取样
取济南南郊菜园通气、湿润、肥沃土壤,用无菌取样铲刮开土壤表层,铲取地表下3-10cm之间的土层放入无菌袋,并在无菌袋上标注取样日期、时间、地点、植被,将所取土壤样品尽快放入4℃冰箱保存,当天至两天内进入菌种富集筛选程序。
(2)筛选
把富集培养液配方中的所有成分(溶菌酶除外)配制成液体培养基,分装,每一250mL三角瓶装入150mL,于121℃高压蒸汽灭菌25min,冷却至25-30℃时,在无菌条件下加入对应量的溶菌酶,混匀。将采集的土样接入配制好的富集培养液中,摇匀,于20-30℃、120-180r/min进行摇瓶培养2-3天,得富集培养菌液。
配制选择性培养基,用Φ9cm平皿制作选择性培养基平板。把富集培养菌液进行系列稀释,选择合适的稀释度涂布选择性培养基平板,30-37℃培养2-5天,挑选褐色或黑色、有粘稠度、有褐色圈的菌落,经纯化,然后挑选典型菌落接种于斜面培养基,培养成熟后于4℃冰箱保存。将筛选到的菌株命名为LHS-m022。
各培养基如下:
富集培养液的组分及用量:葡萄糖20g/L,酵母浸膏2g/L,蛋白胨10g/L,NaCl 5g/L,MgSO4 0.5g/L,溶菌酶(50万U/g)0.03g/L,蒸馏水1000mL。
选择性培养基的组分及用量:葡萄糖20g/L,KNO3 1g/L,K2HPO4 7g/L,MgSO4 0.5g/L,NaCl 0.2g/L,FeSO4 0.0015g/L,CuSO4 0.02g/L,1%K2Cr2O7 5mL,L-酪氨酸5g/L,纯化琼脂粉15g/L。用10%NaOH溶液调pH值为7.0。
斜面培养基的组分及用量:葡萄糖20g/L,玉米浆2g/L,K2HPO4 4g/L,KH2PO4 2g/L,MgSO4 1g/L,FeSO4 0.001g/L。用10%的NaOH溶液调pH值为7.0。
(3)菌株鉴定
筛选的菌株个体形态特征为(如图2所示):幼龄菌丝呈枝状,生长成熟的菌丝基本全部浓缩成串样的细胞。光学显微镜下可以看到幼龄细胞中绿色物质。椭圆分生孢子长轴边5-8μm,短轴边3-4μm。有粘液层,不运动,没有芽孢,好氧。
筛选的菌株的培养特征为(如图1所示):在选择性培养基平板上长成先是脏白色而后黑色、放射状菌落,有光泽、有粘性。培养5天,菌落直径为14.2mm。
筛选菌株的生理生化特征为:同化葡萄糖、果糖、麦芽糖、蔗糖、木糖,并能产酸。不能利用乳糖、糊精、可溶性淀粉、玉米淀粉。能在天门冬素琼脂、伊莫松琼脂上生长,不产生硫化氢。
该菌株的16SrDNA序列测定结果如下(561bp):
GACCTGCGGAAGGATCATTAAAGAGTAAGGGTGCTCAGCGCCCGACCTCCAACCCTTTGTTGTTAAAACTACCTTGTTGCTTTGGCGGGACCGCTCGGTCTCGAGCCGCTGGGGATTCGTCCCAGGCGAGCGCCCGCCAGAGTTAAACCAAACTCTTGTTATTTAACCGGTCGTCTGAGTTAAAATTTTGAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGAGGGGCATGCCTGTTCGAGCGTCATTACACCACTCAAGCTATGCTTGGTATTGGGTGCCGTCCTTAGTTGGGCGCGCCTTAAAGACCTCGGCGAGGCCTCACCGGCTTTAGGCGTAGTAGAATTTATTCGAACGTCTGTCAAAGGAGAGGACTTCTGCCGACTGAAACCTTTATTTTTCTAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAA。
通过菌株形态特征及16SrDNA序列测定结果鉴定菌株LHS-m022为短梗霉(Aureobasidium sp.),该菌株已于2021年7月15日保藏于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),保藏编号为CGMCC No.23050。
实施例2产品的发酵生产及产物的鉴定
1、发酵生产黑色素多糖
液体菌种培养基的组分及用量(g/L):葡萄糖18,玉米浆2.2,K2HPO4 3.8,KH2PO41.5,MgSO4 1,FeSO4 0.002。用10%的NaOH溶液调pH值为7.0。
发酵培养基的组分及用量(g/L):葡萄糖55,玉米浆3.5,K2HPO4 5,KH2PO4 2,MgSO41.2,MnCl2 0.006,FeSO4 0.0015,CuSO4 0.03,L-酪氨酸1。用10%的NaOH溶液调pH值为6.5。
(1)液体菌种培养
将活化培养的斜面菌种接种于液体菌种培养基中,在27℃、170r/min条件下震荡培养22h,得液体菌种备用;
(2)发酵培养
将液体菌种按7%(体积比)的接种量接种于发酵培养基,在摇瓶中进行发酵培养66h,发酵控制条件为:500mL挡板三角瓶分装培养基的量为100mL,温度27℃、搅拌转速为180r/min,发酵结束得到的摇瓶发酵液如图3所示,过滤去除菌体,得到如图4所示的黑色的发酵液(透明液体黑色素多糖),其可溶于水,不溶于乙醇、丁醇、乙醚、丙酮。经测定,其粘度≥200mPa·S,OD300nm≥8,黑色素多糖含量3.0%。
2、发酵产物鉴定
采用柱层析进行纯化后进行鉴定,具体如下:
(1)CTAB法除蛋白
将发酵液(黑色、粘稠)于4℃、10000r/min离心15min,弃沉淀取上清液用CTAB(Cetyltrimethyl-ammonium bromide)法除蛋白。取除去除蛋白含有目标产物的水相(黑色、透明、粘),加1.5倍无水乙醇充分搅匀,5000r/min离心3min,弃上清,对沉淀用6倍无水乙醇充分洗涤两遍,离心,取沉淀在40℃过夜干燥,制成粉状。
(2)离子交换柱和凝胶过滤柱纯化
把CTAB法除蛋白的粉状产物配成10mg/mL的溶液,经DEAE-Sepharose Fast Flow离子交换柱(1.6×40cm玻璃柱,pH7.6 Tris·HCl缓冲体系和0.7mol/L NaCl洗脱液)和Sephadex G200凝胶过滤柱(1.5×60cm玻璃柱,平衡和洗脱液为0.05mol/L NaCl)依次进行纯化,获得纯化发酵产物(黑色)。
(3)FTIR(红外光谱)和HPLC(高效液相)分析
将纯化的发酵产物(黑色)通过KBr压片法进行红外光谱分析,通过高效液相C18柱进行多糖组分分析,红外光谱图分析如图5所示,HPLC分析结果如图6和表1所示。
结果分析:
(1)由于纯化后的发酵产物为黑色的,推测其含有黑色素,产物经过纯化以后,经FTIR分析(如图5所示),参考现有的黑色素的鉴定方法(王岩,陆懋荪,等.几种天然黑色素分子结构的红外光谱表征研究[J].分析试验室,1996,15(6),63-65.),该物质含黑色素的芳香环(1500-1600cm-1附近)、酚羟基(3400cm-1附近强吸收峰)、羧酸(1700cm-1附近峰与3400cm-1附近强吸收峰一起)和多糖的糖环、糖苷、糖环C-H、糖环-OH、糖醛酸等特征性组分基团(≤1500cm-1部分),因此发酵产物判定为:黑色素与多糖的复合物(如果是混合物,经过纯化步骤后会发生分离);
(2)经HPLC分析(如图6和表1所示),该物质的多糖部分含有:葡萄糖、甘露糖、半乳糖、核糖、葡萄糖醛酸和半乳糖醛酸,是典型的生物杂多糖。
因此,鉴定发酵产物为黑色素多糖(黑色素的杂多糖)。
表1检测结果
实施例3:应用性试验
1、絮凝效果
将实施例2制备的液体黑色素多糖用于生物絮凝,取液体黑色素多糖(含量3%)35mL稀释成3倍(含量1%),均等分成4份,分别的处理方式为:①3倍稀释原液;②90℃60min处理;③100℃30min处理;④121℃25min处理。絮凝结果(表2):表明黑色素多糖抗高温,不同的高温处絮凝活性不降低,反而少量提高。显示出了发明产品的罕见、独特的优秀品质。
表2高温处理黑色素多糖的絮凝效果
样品 | ① | ② | ③ | ④ |
OD<sub>550nmm</sub> | 0.097 | 0.095 | 0.082 | 0.083 |
FR(%) | 94.59 | 94.70 | 95.43 | 95.37 |
2、抗氧化性
将实施例2制备的液体黑色素多糖用于抗氧化作用,取液体黑色素多糖(含量3%)5mL,制备成稀释倍数为15、20、25、30、35、40的稀释液各6mL,制得6个样品。其样品的黑色素多糖含量依次为,①0.2%、②0.15%、③0.12%、④0.1%、⑤0.086%、⑥0.075%。根据所述方法测定光吸收A、B、C值。结果(如表3所示)显示黑色素多糖具有很强的抗氧化性,且随样品含量升高抗氧化性逐渐增强。样品含量为0.2%时,抗氧化活性达到99.0%。
表3黑色素多糖的抗氧化活性
样品序号 | ① | ② | ③ | ④ | ⑤ | ⑥ |
黑色素多糖含量(%) | 0.200 | 0.150 | 0.120 | 0.100 | 0.086 | 0.075 |
抗氧化性活性(%) | 99.0 | 79.31 | 41.61 | 38.30 | 31.80 | 29.20 |
3、食品着色
将实施例2制备的液体黑色素多糖用于面团着色,取小麦面粉100g,液体黑色素多糖(含量3%)10mL,水50mL和适量酵母和成面团,经醒发,蒸成黑色有光泽的馒头,如图7所示。若不加酵母,可直接将面团通过面条机加工成黑色有光泽的面条。
SEQUENCE LISTING
<110> 栾兴社
<120> 一株短梗霉及用它制备黑色素多糖的方法
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 561
<212> DNA
<213> 短梗霉(Aureobasidium sp.)LHS-m022的16SrDNA
<400> 1
gacctgcgga aggatcatta aagagtaagg gtgctcagcg cccgacctcc aaccctttgt 60
tgttaaaact accttgttgc tttggcggga ccgctcggtc tcgagccgct ggggattcgt 120
cccaggcgag cgcccgccag agttaaacca aactcttgtt atttaaccgg tcgtctgagt 180
taaaattttg aataaatcaa aactttcaac aacggatctc ttggttctcg catcgatgaa 240
gaacgcagcg aaatgcgata agtaatgtga attgcagaat tcagtgaatc atcgaatctt 300
tgaacgcaca ttgcgcccct tggtattccg aggggcatgc ctgttcgagc gtcattacac 360
cactcaagct atgcttggta ttgggtgccg tccttagttg ggcgcgcctt aaagacctcg 420
gcgaggcctc accggcttta ggcgtagtag aatttattcg aacgtctgtc aaaggagagg 480
acttctgccg actgaaacct ttatttttct aggttgacct cggatcaggt agggataccc 540
gctgaactta agcatatcaa a 561
Claims (10)
1.一株短梗霉(Aureobasidium sp.)LHS-m022,所述菌株的保藏编号为CGMCCNo.23050。
2.权利要求1所述的短梗霉菌株LHS-m022在发酵制备黑色素多糖方面的应用。
3.一种制备黑色素多糖的方法,其特征是,在以葡萄糖为主要成分的发酵培养基中采用权利要求1所述的短梗霉菌株LHS-m022进行发酵,得到含有黑色素多糖的发酵液。
4.如权利要求3所述的一种制备黑色素多糖的方法,其特征是,
所述发酵培养基的组分及用量为:葡萄糖50-65g/L,玉米浆2.5-4.5g/L,K2HPO4 4-7g/L,KH2PO4 1-3g/L,MgSO4 0.8-1.5g/L,MnCl2 0.004-0.009g/L,FeSO4 0.0005-0.003g/L,CuSO4 0.01-0.05g/L,L-酪氨酸0.5-1.5g/L,pH 6.0-7.5。
5.如权利要求4所述的一种制备黑色素多糖的方法,其特征是,
(1)液体菌种培养
将活化培养的斜面菌种接种于液体菌种培养基中,在24-30℃下震荡培养18-28h,得液体菌种备用;
所述液体菌种培养基的组分及用量为:葡萄糖15-25g/L,玉米浆1-3g/L,K2HPO4 2-5g/L,KH2PO4 1-2.5g/L,MgSO4 0.5-1.5g/L,FeSO4 0.0005-0.003g/L,pH6.0-7.5;
(2)发酵培养
将液体菌种按体积比6-10%的接种量接种于发酵培养基,在挡板三角瓶中26-30℃下进行发酵培养66-72h,发酵结束得发酵液,将发酵液过滤去除菌体,得透明液体黑色素多糖。
6.权利要求3-5中任一项所述方法制备的黑色素多糖。
7.权利要求6所述的黑色素多糖作为生物絮凝剂,在地表水、生活污水、工业污水、生态湖水、养殖废水或者发酵培养液絮凝方面的应用。
8.权利要求6所述的黑色素多糖作为食品着色剂,在使食品颜色变黑方面的应用。
9.权利要求6所述的黑色素多糖在生产抗氧化保健品方面,制备有机黑色素多糖铁或者制备有机黑色素多糖钙中的应用。
10.权利要求6所述的黑色素多糖在生产防晒和/或抗氧化的化妆品中的应用。
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