WO2011035530A1 - 黄色素生成缺陷鞘脂单胞菌及其在结冷胶生产中的应用 - Google Patents
黄色素生成缺陷鞘脂单胞菌及其在结冷胶生产中的应用 Download PDFInfo
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- WO2011035530A1 WO2011035530A1 PCT/CN2010/001228 CN2010001228W WO2011035530A1 WO 2011035530 A1 WO2011035530 A1 WO 2011035530A1 CN 2010001228 W CN2010001228 W CN 2010001228W WO 2011035530 A1 WO2011035530 A1 WO 2011035530A1
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- gellan gum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the present invention relates to a strain of Sphingomonas sp. ZD001 (Sphingomonas sp. ZD001) which is defective in yellow pigment production but which is capable of producing normal quality gellan gum and its use in microbial fermentation to prepare gellan gum.
- Gellan Gum is a microbial exopolysaccharide produced by aerobic fermentation of Sphingomonas paucimobilis, which was successfully developed by Kelco in 1978. It is a microbial extracellular polysaccharide which is non-toxic, safe and has good physicochemical properties after xanthan gum.
- Japan was approved for use in food as early as 1988. The United States was approved by the FDA for food in 1992. China approved it as a food thickener and stabilizer in 1996, and more than 10 countries have approved it.
- gellan gum has been widely used as a new type of emulsifier, suspending agent, thickener, stabilizer, gelling agent, slow release agent, film-forming material, etc. in food, medicine, etc. due to its unique and excellent characteristics. In the industrial fields such as chemical industry, the application prospect is broad.
- Gellan gum is composed of Pl,3-D-glucose, pl,4-D-glucuronic acid and al,4-L-rhamnose molar ratio 2:1:1, which is pl,3-D-
- the linking sequence of glucose, Pl, 4-D-glucuronic acid, p-1, 3-D-glucose, al, 4-L-rhamnose constitutes a tetrasaccharide unit.
- An acyl group is attached to the long chain skeleton of the sugar formed by the polymerization of the tetrasaccharide unit, and the molecular weight is generally about 5 x 10 5 - l x 10 6 Daltons.
- Gellan gum is mainly present in two forms, namely a high acyl gellan gum (also known as natural gellan gum) which has not been deacylated, and a low acyl gellan gum which has been artificially deacylated by physicochemical methods.
- There are two acyl groups in the high acyl natural gellan gum namely acetyl and glyceryl groups, usually acetyl attached to the first glucose residue.
- the glyceryl group is attached to the C2 position of the same glucose residue, and generally contains an average of 1 glyceryl group and 0.5 acetyl groups per tetrasaccharide unit.
- the low acyl gellan gum is a product which is substantially free of acyl groups after being subjected to deacylation treatment by a high acyl gellan gum. Therefore, the gellan gum molecules have different molecular weights due to different degrees of deacylation and deacylation. More widely used in the food industry are low acyl gellan gums of relatively low molecular weight.
- the current production of the gellan gum producing bacteria, S. sphaericus also produces the metabolic by-product yellow pigment (mainly carotenoid pigment) during the fermentation process, which not only competes with the gellan gum but also makes the fermentation.
- the liquid turns yellow.
- the amount of ethanol or isopropanol in the decolorization of gellan gum extraction is increased (usually 3 times fermentation broth)
- the volume of ethanol or isopropanol) and the operation time reduce the efficiency of extraction and purification and increase the cost.
- the object of the present invention is to provide a strain of S. sphaericus ZD001 (Sphingomonas sp. ZD001) which is defective in yellow pigment production but which can produce normal quality gellan gum.
- the ZDOO1 strain has been deposited in the China Center for Type Culture Collection. Address: Wuhan University, Wuhan, China, zip code: 430072; preservation date: September 10, 2009; deposit number: CCTCC No:
- ZD001 CTCC NO: M 209198
- the ZD001 (CCTCC NG: M 209198) strain was obtained by isolation and breeding of the inventors of the present application.
- the 16S rDNA was sequenced and molecularly aligned, and its homology with the 16S rDNA nucleotide sequence of Sphingomonas paucimobilis was over 99%.
- the ZD001 (CCTCC NO: M 209198 ) strain was identified as It has been reported that the gellan gum producing strain is a homologous strain of S. sphaericus.
- the 16S rDNA sequence of the ZDOO1 (CCTCC NO: M 209198 ) strain is as follows: AGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCATGCCTAACACATGC AAGTCGAACGAGATCCTTCGGGGTCTAGTGGCGCACGGGTGCGTAACGCGTGG
- the ZD001 (CCTCC NO: M 209198) strain has the following characteristics: Gram-negative bacillus; no spore; straight rod; colony on milk agar plate is milky white; positive oxidase test; positive for contact enzyme test; Oxygen; Decomposes glucose, fructose, xylose, sucrose; positive for starch hydrolysis test; can not liquefy gelatin; does not grow at 43 °C; cell size is 1.5 ⁇ 5, 0 ⁇ 0.8-1.0 ⁇ ; no yellow pigment is produced.
- the invention also relates to the use of the ZD001 (CCTCC NO: M 209198 ) strain for the preparation of gellan gum by microbial fermentation.
- the application is: the ZD001 (CCTCC NO.M 209198) strain is activated by a conventional method, seed cultured, and then inoculated to a fermentation medium conventionally suitable for sphingomonas, 28 ⁇ 32 ° C
- the pH was adjusted to pH 6.8 to 7.2 for 32 to 60 hours to obtain a fermentation broth containing a high acyl gellan gum.
- the fermentation broth can be directly isolated and purified to obtain a high acyl gellan gum, or can be subjected to deacylation treatment to obtain a low acyl gellan gum.
- the method is as follows: the fermentation broth containing the high acyl gellan gum is adjusted to a pH of 5.0 to 6.0, and the temperature is raised to 50 ° C to 70 ° C (preferably 60 ° C ) for 30 min. After ⁇ 2h (preferably 1h), cool down to 40. C ⁇ 50.
- the method is as follows: adding an alkali metal or alkaline earth metal chloride solution (concentration is usually 20%, w/v) to the fermentation broth containing high acyl gellan gum, Adjust pH to 11.0, solid-liquid separation (solid-state separation of plate and frame) to obtain fiber material 1, mix fiber material 1 with water in a volume ratio of 1: 4 ⁇ 6, adjust pH to 2.5 ⁇ 4.0 (preferably pH3.0), wash 15min ⁇ lh (preferably 30min), press-fit to obtain fiber material 2, mix fiber material 2 with water in a volume ratio of 1:9 ⁇ 12, and raise the temperature to 80°C ⁇ 90°C (preferably 83°C ⁇ 87°C) ), adding alkaline reagent to adjust the pH to 9.5 ⁇ 10.5
- the reaction is 8min ⁇ 15min (preferably 10min ⁇ 12min).
- the pH of the reaction solution is neutral, and the filter aid is added (addition amount is 1 ⁇ 3%, w/v, preferably 2%) , filtering, taking the filtrate to the flocculant (preferably 10% by mass, adding volume is 5% of the volume of the filtrate) to flocculate and precipitate, and then separating and drying to obtain the low acyl gellan gum;
- the alkali metal or The alkaline earth metal chloride is one or a mixture of two or more of the following: CaCl 2 , MgCl 2 , NaCl, KC1 (preferably CaCl 2 ), the alkaline agent being one or a mixture of two or more of the following: NaOH, KOH, Na 3 P0 4 , the filter aid is diatomaceous earth, perlite or a mixture thereof, and the flocculant is one or a mixture of two or more of the following: CaCl 2 , MgCl 2 , Na
- the beneficial effects of the invention are mainly embodied in: providing a strain of S. sphaericus ZD001 (CCTCC NO-.M 209198) with yellow pigment deficiency, the fermentation broth is yellow-free and milky white, and can reduce ethanol when extracted. Or the amount of isopropyl alcohol, simplify the process, increase the yield, reduce the cost, and facilitate the industrial production to obtain high-quality gellan gum.
- Example 1 Isolation and identification of ZD001 (CCTCC NO: M 209198 ) strain
- the final concentration of YM agar medium is: 0.30% yeast extract, 0.30% malt extract,
- the concentration of the medium refers to the concentration by mass and volume.
- the concentration of 1% of a component means that lg of the medium contains lg of the substance.
- a strain of ZD001 (CCTCC NO: M 209198) with yellow pigmentation defects was screened.
- the bacterium is Gram-negative bacillus; no spore; straight rod; colony on milk agar plate is milky white; oxidase test positive; contact enzyme test positive; obligate aerobic; decomposition of glucose, fructose, xylose, sucrose; Starch hydrolysis test was positive; gelatin could not be liquefied; no growth at 43 °C; cell size 1.5-5.0 ⁇ ⁇ 0.8-1. ⁇ ; no yellow pigment.
- Example 2 Fermentation process of ZD001 (CCTCC NO: M 209198)
- the pure strain is inoculated on the inclined surface of YPG medium and cultured at 30 ° C for 72 hours;
- the slant seed is connected to a 50 mL-grade seed culture medium (in a 250 mL flask) and shaken at 30 ° C, 200 rpm for 24 h, which is a first-class seed liquid;
- Secondary seed culture The first-stage seed liquid is connected to 100 mL of secondary seed culture medium (in a 500 mL flask) at a concentration of 5% by volume, and cultured at 30 ° C, 200 rpm for 12 h, which is the second stage. Seed liquid
- the secondary seed liquid is connected to the fermentation medium at a dose of 5% by volume, and the mixture is ventilated at 0 °C, pH 6.8 -7.2, at a speed of 300 rpm for 48 hours;
- composition and final concentration of YPG slant medium were: 2.00% glucose, 0.50% peptone, Yeast extract 0.30%, agar 1.50%, solvent is distilled water, pH 7.2;
- the final concentration of the first-stage seed medium is: yeast extract 0.20%; beef extract 0.30%; protein ⁇ 0.50%; potassium chloride 0.10%, solvent is distilled water, pH 7.2;
- Secondary seed culture medium glucose 1.50%; yeast extract. 0.50%; peptone 0.50%; potassium dihydrogen phosphate 0.06%; dipotassium hydrogen phosphate 0.06%; magnesium sulfate 0.06%, solvent distilled water, pH 7.2; fermentation medium : glucose 3.00%; yeast extract 0.05%; peptone 0.30%; potassium dihydrogen phosphate 0.06%; dipotassium hydrogen phosphate 0.10%; magnesium sulfate 0.06%; solvent distilled water, pH 7.2.
- Example 3 Preparation process of high acyl gellan gum
- Fermentation liquid pretreatment 100 L of fermentation broth, adjusted to pH 6.0 with 10% (v/v) of HC1, heated to 60 ° C, and kept for 1 h;
- Pretreatment of fermentation broth Take 100L of mature fermentation broth, add 3L of CaCl 2 solution with concentration of 20% (w/v), stir lOmin ⁇ , adjust pH to 11.0 with 10% NaOH, stir for 3min ⁇ plate filter press, get Fiber material 2. Washing: The fiber material pretreated in step 1 is mixed with water in a volume ratio of 1:5, adjusted to pH 3.0 with 10% HCl, stirred for 30 minutes, and framed by pressure filtration to obtain a fiber material.
- Deacylation treatment Mix the fiber material washed in step 2 with water in a volume ratio of 1:10, stir evenly, and raise the temperature to about 90 , to completely dissolve the fiber material, maintain the temperature at 85 ° C, add 10%.
- NaOH aqueous solution adjusted pH 9.5 ⁇ 10.5, reaction 10min ⁇ , adjusted to pH6.0 with 10% HC1;
- Drying and pulverizing The cold gelatin fiber material obtained by pressing is dried at 90 ° C for 2 h ⁇ pulverization to obtain a finished product of low acyl gellan gum.
- the resulting low acyl gellan gum is a milky white powder having a strength > 800 g/cm 2 and a light transmittance > 80%.
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Description
说 明 书
黄色素生成缺陷鞘脂单胞菌及其在结冷胶生产中的应用
(一 )技术领域
本发明涉及一株黄色素生成缺陷但能生产正常品质结冷胶的鞘脂单 胞菌菌株 ZD001 ( Sphingomonas sp. ZD001 )及其在微生物发酵制备结冷 胶中的应用。
(二)技术背景
结冷胶 (Gellan Gum)是少动鞘脂单胞菌 ( Sphingomonas paucimobilis )经好氧发酵产生的微生物胞外多糖, 由美国 Kelco公司于 1978年开发成功。 它是继黄原胶之后又一种无毒、 安全、 理化性质优良 的微生物胞外多糖。 日本早在 1988年就准许在食品中使用, 美国于 1992 年获 FDA批准而用于食品, 我国于 1996年批准其作为食品增稠剂、 稳 定剂使用, 另有十多个国家也已批准用作食品添加剂。 近年来, 结冷胶以 其独特的优良特性,被作为新型乳化剂、 悬浮剂、 增稠剂、 稳定剂、 凝胶 剂、緩释剂、成膜材料等日益广泛地应用于食品、 医药、化工等工业领域, 应用前景广阔。
结冷胶是由 P-l,3-D-葡萄糖, p-l,4-D-葡萄糖醛酸和 a-l,4-L-鼠李糖按 摩尔比 2: 1: 1组成,其以 p-l,3-D-葡萄糖、 P-l,4-D -葡萄糖醛酸、 p_l,3-D- 葡萄糖、 a-l,4-L -鼠李糖的连接顺序构成四糖单元。 在四糖单元聚合形成 的糖长链骨架上连有酰基, 分子量一般为 5x l05 - l x lO6道尔顿左右。 结冷 胶主要以两种形式存在,即未经脱酰基的高酰基结冷胶(也称天然结冷胶) 和经物理化学方法人工脱酰基的低酰基结冷胶。高酰基天然结冷胶中有两 种酰基, 即乙酰基和甘油酰基, 通常乙酰基连接在第一个葡萄糖残基的
C6位上, 而甘油酰基连接在同一个葡萄糖残基的 C2位上, 一般每个四糖 单元中平均含有 1个甘油酰基, 0.5个乙酰基。 低酰基结冷胶则是由高酰基 结冷胶通过脱酰基处理后而基本上不含酰基的产物, 因此,结冷胶分子因 是否脱酰基和脱酰基程度不同,分子量差异较大, 目前在食品工业上应用 较多的是分子量相对较低的低酰基结冷胶。
当前生产所用的结冷胶产生菌少动鞘脂单胞菌在发酵过程中同时会 产生代谢副产物黄色素 (主要是类胡萝卜色素), 这不仅与结冷胶竟争碳 源, 还使发酵液变黄。在结冷胶制备过程中, 尤其是在高酰基结冷胶的制 备过程中,为了去除胶体中的黄色素,增加了结冷胶提取脱色中乙醇或异 丙醇的用量(一般用 3倍发酵液体积的乙醇或异丙醇)与操作时间,使提 取纯化的效率降低、 成本提高。
(三)发明内容 本发明的目的是提供一株黄色素生成缺陷但能生产正常品质结冷胶 的鞘脂单胞菌菌株 ZD001 ( Sphingomonas sp. ZD001 )。 该 ZDOOl菌株已 保藏于中国典型培养物保藏中心, 保藏中心地址: 中国 武汉 武汉大学, 邮政编码: 430072;保藏日期: 2009年 9月 10日;保藏编号: CCTCC No:
M209198。 故该菌株以下称 ZD001 ( CCTCC NO: M 209198 ) 菌株。
本发明采用的技术方案:
ZD001 ( CCTCC NG:M 209198 )菌株是本申请发明人分离选育所得。 对其 16S rDNA 进行测序与分子比对, 其与少动鞘脂单胞菌 ( Sphingomonas paucimobilis ) 16S rDNA核苷酸亭列同源性为 99%以上, 认定 ZD001 ( CCTCC NO: M 209198 )菌株为已见报道的结冷胶产生菌少 动鞘脂单胞菌的同种变异株。
所述 ZDOOl ( CCTCC NO: M 209198 ) 菌株的 16S rDNA序列如下: AGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCATGCCTAACACATGC AAGTCGAACGAGATCCTTCGGGGTCTAGTGGCGCACGGGTGCGTAACGCGTGG
ATGTCGAAAGACCAAAGATTTATCGCCCTGAGATGAGCCCGCGTAGGATTAGCT AGTTGGTGTGGTAAAGGCGCACCAAGGCGACGATCCTTAGCTGGTCTGAGAGG
AGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGAGT
CGGGAGAATAAGCCCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGG GGGCTAGCGT GTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGCTTTGTA
GATATTCGGAAGAACACCAGTGGCGAAGGCGGCTCACTGGACTGGTATTGACGC
TGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACG
CCGTAAACGATGATAACTAGCTGTCCGGGTGCTTGGCACTTGGGTGGCGCAGCT
AATTGACGGGGGCCTGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAAC
CTTCCCTTCGGGGACCTACACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTC
TCATTAAGTTGGGTACTTTAAAGTAACCGCCGGTGATAAGCCGGAGGAAGGTGG
GGATGACGTCAAGTCCTCATGGCCCTTACGCGCTGGGCTACACACGTGCTACAA
TGGCAAGTACAGTGGGCAGCAATCCCGCGAGGGTGAGCTAATCTCCAAAACTT
GTCTCAGTTCGGATTGTTCTCTGCAACTCGAGAGCATGAAGGCGGAATCGCTAG
TAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCAGGCCTTGTACACACCG
CCCGTCACACCATGGGAGTTGGGTTCACCCGAAGGCGTTGCGCTAACTCGTAAG
AGAGGCAGGCGACCACGGTGGGCTTAGCGACTGGGGTGAAGTCGTAACAAGGT
AGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTo
所述 ZD001 ( CCTCC NO:M 209198 ) 菌株特征如下: 为革兰氏阴性 杆菌;无芽孢;直杆状;在营养琼脂平板上菌落呈乳白色;氧化酶试验阳性; 接触酶试验阳性; 专性需氧; 分解葡萄糖、 果糖、 木糖、 蔗糖; 淀粉水解 试验阳性;不能液化明胶;43°C不生长;菌体大小为 1.5~5,0μπιχ0.8-1.0μπι; 不产黄色素。
本发明还涉及所述的 ZD001 (CCTCC NO: M 209198 ) 菌株在微生物 发酵制备结冷胶中的应用。
具体的, 所述应用为: 将所述 ZD001 (CCTCC NO.M 209198 ) 菌株 按常规方法经活化、种子培养后,接种至常规适用于鞘脂单胞菌的发酵培 养基, 28~32°C、 pH6.8〜7.2摇床培养 32~60h, 获得含有高酰基结冷胶的 发酵液。该发酵液可直接分离纯化得到高酰基结冷胶,也可进行脱酰基处 理制取低酰基结冷胶。
制备产物为高酰基结冷胶时,方法如下:将所述含有高酰基结冷胶的 发酵液调 pH值为 5.0~6.0,升温至 50°C~70°C (优选 60 °C )保温 30min~2h (优选 lh )后, 降温至 40。C~50。C , 调 pH为 7.0, 加入溶菌酶和碱性蛋 白酶保温 lh~3h (优选 2h )去除蛋白质; 去除蛋白质后的发酵^ σ入絮 凝剂溶液进行絮凝沉淀 (絮凝剂溶液加入量约为去除蛋白质后发酵液体积 的 5% ), 再经分离 (加入体积为发酵液体积 30%左右的体积浓度 90%以 上的异丙醇或乙醇, 优选 95%异丙醇, 搅拌, 板框压滤)、 干燥(90°C ), 制得所述高酰基结冷胶, 所述絮凝剂 (浓度通常为 20%, w/v )为下列之 一或其中两种以上的混合: CaCl2、 MgCl2、 NaCl、 KC1。
制备产物为低酰基结冷胶时,方法如下:往所述含有高酰基结冷胶的 发酵液中加入碱金属或碱土金属氯化物溶液(浓度通常为 20%, w/v ),
调 pH为 11.0, 固液分离 (板框固液分离)得到纤维料 1, 将纤维料 1与 水按 1: 4〜6体积比混合,调 pH2.5~4.0 (优选 pH3.0 ), 洗涤 15min~lh (优 选 30min ), 压滤得到纤维料 2, 将纤维料 2与水按 1: 9~12体积比混合 均匀 ,升温至 80°C~90°C(优选 83 °C~87°C ),加入碱性试剂调 pH为 9.5~10.5
(优选 pH9.8~10.2 )反应 8min~15min (优选 10min~12min ), 反应结束后 调反应液 pH为中性, 加入助滤剂(添加量为 1~3%, w/v, 优选 2% ), 过 滤,取滤液加入絮凝剂(优选 10%质量浓度,添加体积为滤液体积的 5% ) 进行絮凝沉淀, 再经分离、 干燥, 制得所述低酰基结冷胶; 所述碱金属或 碱土金属氯化物为下列之一或其中两种以上的混合: CaCl2、MgCl2、NaCl、 KC1 (优选 CaCl2), 所述碱性试剂为下列之一或其中两种以上的混合: NaOH、 KOH、 Na3P04, 所述助滤剂为硅藻土、 珍珠岩或其混合物, 所述 絮凝剂为下列之一或其中两种以上的混合: CaCl2、 MgCl2、 NaC KC1
(优选 KC1)。
本发明的有益效果主要体现在:提供了一株黄色素生成缺陷的鞘脂单 胞菌 ZD001 ( CCTCC NO-.M 209198 ) 菌株, 其发酵液不含黄色素而呈乳 白色,提取时可减少乙醇或异丙醇用量,简化工序,提高得率,降低成本, 利于工业化生产获得优质的结冷胶。
(四)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围 并不仅限于此:
实施例 1 : ZD001 ( CCTCC NO: M 209198 ) 菌株的分离及鉴定
1、 无菌采集土壤样品, 稀释涂布于 YM琼脂培养基平板上;
2、 该平板置于 30°C培养 5天;
3、 挑取不同形态的细菌菌落, 于相同培养基平板分别划线纯化分离, 置 于 30°C培养 5天。
YM琼脂培养基终浓度组成为: 0.30%酵母抽提物, 0.30%麦芽抽提物,
0.50、蛋白胨, 1.00%葡萄糖, 琼脂 1.50% , 溶剂为蒸馏水。
注: 本发明中培养基浓度均指质量体积百分比浓度, 某组分浓度 1%表示 lOOmL培养基中含有 lg该物质。
经筛选获得一株黄色素生成缺陷的 ZD001 ( CCTCC NO:M 209198 ) 菌株。 该菌为革兰氏阴性杆菌;无芽孢;直杆状; 在营养琼脂平板上菌落呈 乳白色; 氧化酶试验阳性; 接触酶试验阳性; 专性需氧; 分解葡萄糖、 果 糖、 木糖、 蔗糖; 淀粉水解试验阳性;不能液化明胶; 43°C不生长; 菌体 大小为 1.5-5.0μπι χ 0.8-1. Ομιη; 不产黄色素。 实施例 2: ZD001 ( CCTCC NO: M 209198 )的发酵工艺
1、 纯菌种接种于 YPG培养基斜面上, 30°C培养 72h;
2、 一级种子培养: 将斜面种子接入 50mL—级种子培养基(盛于 250mL 三角瓶), 于 30°C, 200rpm振荡培养 24h, 即为一级种子液;
3、 二级种子培养: 将一级种子液以 5%体积比的接种量接入 lOOmL二级 种子培养基(盛于 500mL三角瓶), 于 30°C, 200rpm振荡培养 12h, 即 为二级种子液;
4、 罐上发酵: 将二级种子液以 5%体积比的接种量接入发酵培养基中, 30°C、 pH 6.8 -7.2条件下, 通风 1. 0 m, 转速 300rpm, 培养 48小时;
5、 收集发酵产物: 收集步骤 4培养 48小时的含结冷胶发酵液。
YPG斜面培养基组成与终浓度为: 葡萄糖 2.00%, 蛋白胨 0.50%,
酵母抽提物 0.30%, 琼脂 1.50% , 溶剂为蒸镏水, pH7.2;
一级种子培养基终浓度组成为: 酵母膏 0.20%; 牛肉膏 0.30%; 蛋白 胨 0.50%; 氯化钾 0.10%, 溶剂为蒸馏水, pH7.2;
二级种子培养基: 葡萄糖 1.50%; 酵母膏 .0.50%; 蛋白胨 0.50%; 磷 酸二氢钾 0.06%;磷酸氢二钾 0.06%;硫酸镁 0.06%,溶剂为蒸馏水, pH7.2; 发酵培养基: 葡萄糖 3.00%; 酵母膏 0.05%; 蛋白胨 0.30%; 磷酸二 氢钾 0.06%; 磷酸氢二钾 0.10%; 硫酸镁 0.06%; 溶剂为蒸馏水, pH7.2。 实施例 3: 高酰基结冷胶的制备工艺
1、发酵液预处理:发酵液 100L,用 10%( v/v )的 HC1调 pH6.0,升温至 60°C , 保温 lh;
2、蛋白杂质去除: 降温至 40°C时, 用 10% ( w/v ) NaOH调 pH7.0, 加入 50g 的溶菌酶(20万 U/g, 庞博生物)及 100g的碱性蛋白酶(2万 U/g, 庞博生 物), 保温 2h;
3、 絮凝沉淀, 分离: 经过预处理及蛋白去杂处理后的发酵液中加入 5L浓 度为 20% ( w/v ) 的 CaCl2 溶液, 搅拌 30min后, 加入 30L异丙醇, 继续搅 拌 lh后, 板框压滤;
4、 干燥、 粉碎: 压滤所得纤维料 90°C干燥 2h后粉碎, 制得高酰基结冷 胶成品, 为乳白色粉末, 含氮量为 0.05~0.30%。 实施例 4: 脱酰结冷胶的制备工艺
1、发酵液预处理: 取成熟发酵液 100L,加入 3L浓度为 20% ( w/v )的 CaCl2 溶液, 搅拌 lOmin^用 10% NaOH调 pH至 11.0, 搅拌 3min^板框压滤, 得 纤维料;
2、洗涤:将步骤 1预处理后的纤维料与水按 1: 5的体积比混合,用 10%HC1 调 pH3.0, 搅拌 30min^板框压滤, 得纤维料。
3、 脱酰基处理: 将步骤 2洗'涤后的纤维料与水按 1: 10的体积比混合, 搅 拌均匀, 升温至 90Ό左右, 使纤维料完全溶解, 维持温度 85°C, 加入 10% NaOH水溶液, 调 pH值 9.5〜10.5, 反应 10min^, 用 10%HC1调 pH6.0;
4、加入盾量为料液体积 2%的硅藻土, 维持温度 80°C以上板框过滤后加入 料液(滤过液)体积 5%的 KC1溶液(质量浓度为 10% ), 板框压榨;
5、 干燥、 粉碎: 压榨所得结冷胶纤维料 90°C干燥 2h^粉碎, 制得低酰基 结冷胶成品。 所得的低酰基结冷胶为乳白色粉末, 强度 > 800 g/cm2, 透 光率 > 80%。
Claims
1. 一株黄色素生成缺陷鞘脂单胞菌 Sphingomonas sp. ) ZD001 , 保藏于 中国典型培养物保藏中心, 地址: 中国 武汉 武汉大学, 430072, 保 藏日期: 2009年 09月 10日, 保藏编号: CCTCC No: M 209198。
2. 如权利要求 1 所述的鞘脂单胞菌, 其特征在于所述鞘脂单胞菌的 16S rDNA序列如下:
AGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCATGCCTAACACATGCA
AGTCGAACGAGATCCTTCGGGGTCTAGTGGCGCACGGGTGCGTAACGCGTGG
GAATCTGCCTTGGGGTTCGGAATAACTCCCCGAAAGGGGTGCTAATACCGGA
GCTAGTTGGTGTGGTAAAGGCGCACCAAGGCGACGATCCTTAGCTGGTCTGA
GAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGA
GGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCAATGCC
GCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTTACCCGGGAAGATA
ATGACTGTACCGGGAGAATAAGCCCCGGCTAACTCCGTGCCAGCAGCCGCGG
TAATACGGAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGT
AGGCGGCTTTGTAAGTCAGAGGTGAAAGCCTGGAGCTCAACTCCAGAACTG
CCTTTGAGACTGCATCGCTTGAATCCAGGAGAGGTGAGTGGAATTCCGAGTG
TAGAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGGCGAAGGCGGCTC
ACTGGACTGGTATTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGAT
TAGATACCCTGGTAGTCCACGCCGTAAACGATGATAACTAGCTGTCCGGGTGC
TTGGCACTTGGGTGGCGCAGCTAACGCATTAAGTTATCCGCCTGGGGAGTAC
GGCCGCAAGGTTAAAACTCAAAGGAATTGACGGGGGCCTGCACAAGCGGTG
GAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCGTTTGACA
TGGTAGGACGACTGGCAGAGATGCCTTTCTTCCCTTCGGGGACCTACACACA
GGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC
GCAACGAGCGCAACCCTCGACTTTAGTTACCATCATTAAGTTGGGTACTTTAA
AGTAACCGCCGGTGATAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCA TGGCCCTTACGCGCTGGGCTACACACGTGCTACAATGGCAAGTACAGTGGGC
AGCAATCCCGCGAGGGTGAGCTAATCTCCAAAACTTGTCTCAGTTCGGATTG
TTCTCTGCAACTCGAGAGCATGAAGGCGGAATCGCTAGTAATCGCGGATCAG
CATGCCGCGGTGAATACGTTCCCAGGCCTTGTACACACCGCCCGTCACACCAI
GGGAGTTGGGTTCACCCGAAGGCGTTGCGCTAACTCGTAAGAGAGGCAGGC
GACCACGGTGGGCTTAGCGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAG
GGGAACCTGCGGCTGGATCACCTCCTT。
3. 如权利要求 1 所述的鞘脂单胞菌, 其特征在于所述鞘脂单胞菌菌株特 征如下: 为革兰氏阴性杆菌;无芽孢;直杆状; 在营养琼脂平板上菌落呈 乳白色; 氧化酶试验阳性; 接触酶试验阳性; 专性需氧; 分解葡萄糖、 果糖、 木糖、 蔗糖; 淀粉水解试验阳性; 不能液化明胶; 43°C不生长; 菌体大小为 1.5〜5.0μπιχ0.8-1.0μιη; 不产黄色素。
4. 如权利要求 1所述的鞘脂单胞菌在微生物发酵制备结冷胶中的应用。
5. 如权利要求 4所述的应用, 其特征在于所述应用为: 将所述鞘脂单胞 菌菌林经活化、 种子培养后, 接种至适用于鞘脂单胞菌的发酵培养基, 28~32°C pH6.8~7.2摇床培养 32~60h, 获得含有高酰基结冷胶的乳白 色发酵液。
6. 如权利要求 5 所述的应用, 其特征在于所述含有高酰基结冷胶的发酵 液经分离纯化, 制得高酰基结冷胶; 所述分离纯化方法如下: 含有高 酰基发酵液调 pH值为 5.0 6.0, 升温至 50°C~70°C保温 30min~2h后, 降温至 40°C~50°C,调 pH为 7.0,加入溶菌酶和碱性蛋白酶保温 lh〜3h 去除蛋白质; 去除蛋白质后的发酵液加入絮凝剂溶液进行絮凝沉淀, 再经分离、 干燥, 制得所述高酰基结冷胶, 所述絮凝剂为下列之一或 其中两种以上任意比例的混合: CaCl2、 MgCl2、 NaCK KC1。
7. 如权利要求 5 所述的应用, 其特征在于所述含有高酰基结冷胶的发酵 液经脱酰基处理后、 再经分离纯化制得低酰基结冷胶, 所述的低酰基 结冷胶的制备方法如下: 含有高酰基结冷胶的发酵液加入碱金属或碱 土金属氯化物溶液, 调 pH为 11.0, 固液分离得到纤维料 1, 将纤维料 1与水按 1: 4~6体积比混合, 调 pH2.5~4.0, 洗涤 15min~lh, 压滤得 到纤维料 2, 将纤维料 2 与水按 1 : 9-12体积比混合均匀, 升温至 80°C~90°C , 加入碱性试剂调 pH为 9.5~10.5反应 8min~15min, 反应结 束后调反应液 pH为中性, 加入助滤剂, 过滤, 取滤液加入絮凝剂进行 絮凝沉淀, 再经分离、 干燥, 制得所述低酰基结冷胶; 所述碱金属或 碱土金属氯化物为下列之一或其中两种以上的混合: CaCl2、 MgCl2、 NaCl、 KC1, 所述碱性试剂为下列之一或其中两种以上的混合: NaOH、 KOH、 Na3P04, 所述助滤剂为硅藻土、 珍珠岩或其混合, 所述絮凝剂 为下列之一或其中两种以上的混合: CaCl2、 MgCl2、 NaCl、 KC1。
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CN102311508B (zh) * | 2010-07-09 | 2014-05-14 | 郸城财鑫糖业有限责任公司 | 一种透明型高酰基结冷胶的提取工艺 |
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CN103509845B (zh) * | 2013-08-09 | 2015-07-08 | 新疆阜丰生物科技有限公司 | 一种高酰基结冷胶的提取方法 |
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