WO2007137510A1 - Souche de bacillus pumilus destinée à produire un rendement élevé de tétraméthylpyrazine - Google Patents

Souche de bacillus pumilus destinée à produire un rendement élevé de tétraméthylpyrazine Download PDF

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WO2007137510A1
WO2007137510A1 PCT/CN2007/001705 CN2007001705W WO2007137510A1 WO 2007137510 A1 WO2007137510 A1 WO 2007137510A1 CN 2007001705 W CN2007001705 W CN 2007001705W WO 2007137510 A1 WO2007137510 A1 WO 2007137510A1
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tetramethylpyrazine
bacillus pumilus
strain
fermentation
cctcc
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PCT/CN2007/001705
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English (en)
French (fr)
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Ping Xu
Zijun Xiao
Zhonghao Wei
Yi Du
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Shanghai Apple Flavor & Fragrance Co., Ltd
Shanghai Kaixin Biotech Co., Ltd
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Application filed by Shanghai Apple Flavor & Fragrance Co., Ltd, Shanghai Kaixin Biotech Co., Ltd filed Critical Shanghai Apple Flavor & Fragrance Co., Ltd
Priority to AT07721278T priority Critical patent/ATE485364T1/de
Priority to DE602007009983T priority patent/DE602007009983D1/de
Priority to US12/302,275 priority patent/US8067212B2/en
Priority to EP07721278A priority patent/EP2031052B1/en
Publication of WO2007137510A1 publication Critical patent/WO2007137510A1/zh

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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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12R2001/07—Bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus

Definitions

  • the present invention is in the field of biotechnology, and in particular relates to a newly isolated, highly productive tetramethylpyrazine-producing Bacillus pumilus. Background technique
  • Tetramethylpyrazine (2, 3, 5, 6-tetramethylpyrazine) was first isolated from Ligusticim ⁇ i i in 1957. Tetramethylpyrazine has aromas such as cocoa, coffee, lactose, wine, gems, etc. It is widely used in the fragrance industry of food, cigarettes, alcohol and so on. Tetramethylpyrazine is also an active alkaloid that plays a major role in the traditional Chinese medicine Chuanxiong. In recent years, with the deepening of its research, people have continuously discovered its valuable value in pharmacy. It has been widely used as a major drug for diseases of circulation, central nervous system, respiratory and other system diseases, and is widely used in cardiovascular and cerebrovascular diseases and respiratory system. Treatment of diseases, glomerular diseases, etc.
  • the production method of tetramethylpyrazine can be divided into three methods: biosynthesis, direct extraction from plants, and chemical synthesis.
  • bioengineering technology mainly refers to microbial engineering technology, including strain screening and mutagenesis technology and fermentation engineering technology to produce tetramethylpyrazine.
  • microbial engineering technology including strain screening and mutagenesis technology
  • fermentation engineering technology to produce tetramethylpyrazine.
  • Tetramethylpyrazine can be obtained by fermentation of various sugars. The research status of biosynthesis of tetramethylpyrazine is described below.
  • Itohiki-natto is one of the most popular fermented foods due to its unique pyrazine flavor. It is produced by the Japanese using a microorganism called Bacillus natto) on soybean solid medium. However, since the solid fermentation uses mainly empirical methods, the fermentation results are less than satisfactory, and the resulting perfume compound yields a wet weight of no more than 22 ⁇ g/kg (Kosuge et al., 1971).
  • the method for extracting tetramethylpyrazine from plants has limited source of raw materials, and the content of tetramethylpyrazine in the raw materials is too low, the extraction cost is too high, and industrialization cannot be achieved; chemical synthesis methods generally have serious environmental problems, and reactions The conditions are generally severe, the equipment requirements are higher, and the product is not natural; biosynthesis has the outstanding advantages of green natural product, low cost, mild reaction conditions, and low environmental pollution. It is a future research on the synthesis of tetramethylpyrazine.
  • the purpose of the present invention is to provide a newly isolated technical solution for the production of tetramethylpyrazine, and to provide a newly isolated DNA-producing tetramethylpyrazine-producing Bacillus pumilus which can be fermented to produce tetramethylpyrrol. Oxazine.
  • the Bacillus pumilus RX3-17 which is referred to in the present invention, has been deposited with the China Center for Type Culture Collection on April 19, 2006, and has a deposit number of CCTCC M 206043.
  • the SV fermentation medium is formulated as follows: 1 liter of distilled water containing: 200 g of glucose, 50 g of soy peptone, 30 g of (NH 4 ) 2 HP 0 4 , 15 ml of a vitamin mixture.
  • the composition of the vitamin mixture is: 1 liter of distilled water containing: 1. 0 mg of biotin, 1. 0 mg of vitamin B 1 5. 0 mg of vitamin B 2 , 25 mg of vitamin ⁇ ⁇ , 50 mg of p-aminobenzoic acid, 100 Mg millicalcium pantothenate, 100 mg niacin.
  • Bacillus pumilus RX3-17 CCTCC M 206043 strain used to ferment tetramethylpyrazine in SV fermentation medium, the fermentation temperature is 37 ° C, 50 ml of fermentation broth is placed in a 500 ml Erlenmeyer flask, placed The shaken bed was incubated at 120 rpm for 96 to 120 hours to obtain a mature tetramethylpyrazine fermentation broth.
  • the high-yield tetramethylpyrazine-producing Bacillus pumilus provided by the present invention solves the bottleneck problem of low concentration of fermentation products in the production of tetramethylpyrazine by microbial fermentation.
  • the production of tetramethylpyrazine by microbial fermentation has the advantages of low cost and easy availability of raw materials, mild reaction conditions, green and natural products, low cost and environmental friendliness.
  • the Bacillus pumilus RX3-17 which is referred to in the present invention, has been deposited with the China Center for Type Culture Collection on April 19, 2006, and has a deposit number of CCTCC M 206043.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an enlarged 2700-fold electron micrograph of the Bacillus pumilus RX3-17 CCTCC M 206043 strain of the present invention. , . detailed description
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • Step 1 Mutagenesis screening of Bacillus pumilus RX3-17 CCTCC M 206043 strain:
  • Soil samples were taken from apple orchards, vineyards, etc., suspended in physiological saline, heated under 80 Torr for 10 minutes, and then streaked onto a nutrient agar dish and cultured at 37 °C.
  • the nutrient agar medium formula is: 1 liter of distilled water containing: peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 18 g.
  • the initial pH of the medium was 7.2, the sterilization temperature was 121 ⁇ , and the sterilization time was 15 minutes.
  • the single bacteria were picked out, inoculated in a nutrient broth medium, and cultured under a 37 C condition on a shaker at 120 rpm. 5 ⁇ 30 After treatment to a long period of logarithmic growth, the cells were suspended in 0. 05 mol / liter of Tris-maleic acid buffer (pH 6. 0), treated with 0.5 mg / liter of nitrosoguanidine 30 minute. After the mutagenesis process was completed by the dilution method, the mutagenized culture was placed on a shaker for 5 hours, and then appropriately diluted and spread on a nutrient agar dish for further screening.
  • the preparation method of the nutrient broth medium is the same as the preparation method of the above nutrient agar medium except that no agar is added.
  • the physiological and biochemical identification experiments showed that the morphology of the strain obtained by the above experiment was a rod-shaped, long 1. 5 ⁇ 3. 0 micron, diameter 0. 6 ⁇ 0. 7 microns, colony color is milky white, spore-forming, VP reaction Positive, available glucose, arabinose, xylose, mannitol, fructose acid, hydrolyzable casein, gelatin, Tween 80, available citrate, available in The mass percentage is 10% NaCl growth, can be grown at 50 ° C, and its 16S rDNA sequence is 99% similar to the other 16S rDNA sequences of Bacillus pumilus.
  • the strain was named Bacillus pumilus RX3-17 and was deposited with the China National Type Culture Collection on April 19, 2006. The deposit number is CCTCC M 206043.
  • Step 2 Preparation of a cell culture of the above Bacillus pumilus RX3-17 CCTCC M 206043 strain: Picking a slant culture with a nutrient agar medium pumilus RX3-17 CCTCC M 206043 a ring strain, was inoculated in 25 ml of nutrient broth medium containing sterilized 300 ml conical flask at 37 a C, 120 rpm / min incubation on a shaker for 12 hours That is, a cell liquid culture of the ciL pumilus RX3-17 CCTCC M 206043 strain was prepared.
  • Step 3 Using the above Bacillus pumilus RX3-17 CCTCC M 206043 strain, a mature tetramethylpyrazine fermentation broth was obtained from an SV fermentation medium containing 160 g/L of glucose:
  • the cell liquid culture of the Bacillus pumilus RX3-17 CCTCC M 206043 strain obtained by the method of Example 2 was inoculated at a volume percentage of 6% inoculum to a 500 ml cone containing sterilized 50 ml SV fermentation medium. In a flask, culture on a shaker at 37 Torr, 120 rpm. At the 96th hour, the concentration of tetramethylpyrazine reached 1.50 ⁇ 0.07 g / liter, which gave a mature tetramethylpyrazine fermentation broth.
  • the above SV fermentation medium containing 160 g/L of glucose is formulated as follows: 1 liter of distilled water containing: 160 g of glucose, 50 g of soy peptone, 30 g of (NH 4 ) 2 HP0 4 , 15 ml of vitamin mixture, adjusting ⁇ to 7 , 0.
  • the composition of the vitamin mixture is: 1 liter of distilled water containing: 1. 0 mg of biotin, 1. 0 mg of vitamin ⁇ , 5. 0 mg of vitamin ⁇ 2 , 25 mg of vitamin ⁇ 6 , 50 mg of p-aminobenzoic acid, 100 mg Calcium pantothenate, 100 mg niacin.
  • Example 2 Bacillus pumilus RX3-17 CCTCC M 206043 strain obtained by the method of the above-mentioned Example 1 steps 1 and 2, a mature tetramethylpyrazine fermentation broth was obtained from an SV fermentation medium containing 180 g/L of glucose: The same as step 3 of the first embodiment described above, except that the glucose concentration was changed to 180 g/liter. O. 08 ⁇ / ⁇ The concentration of the tetramethylpyrazine was 1. 79 ⁇ 0. 08 g / liter.
  • Example 3 Bacillus pumilus RX3-17 CCTCC 206043 strain obtained by the method of the above-mentioned Example 1 steps 1 and 2, a mature tetramethylpyrazine fermentation broth was obtained from an SV fermentation medium containing 200 g/L of glucose: Step 3 of Example 1 was the same except that the glucose concentration was changed to 200 g/liter. O. 06 ⁇ / ⁇ The broth was obtained at the end of the day, the concentration of tetramethylpyrazine reached 2. 22 ⁇ 0. 06 g / liter.
  • Example 4 Using the above Bacillus pimilus Wi-VI CCTCC M 206043 strain obtained by the method of the above-mentioned Example 1 steps 1 and 2, a mature tetramethylpyrazine fermentation broth was obtained from a SV fermentation medium containing 220 g/L of glucose. :
  • step 3 of the first embodiment The same as step 3 of the first embodiment, except that the glucose concentration was changed to 220 g / liter. At the 120th hour, a mature fermentation broth was obtained, wherein the concentration of tetramethylpyrazine reached 1.67 ⁇ 0.05 g / liter.

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Description

一株高产四甲基吡嗪的短小芽孢杆菌 技术领域
本发明属于生物技术领域， 具体地说涉及一株新分离的高产四甲基吡嗪的短小芽孢 杆菌。 背景技术
四甲基吡嗪（2, 3, 5, 6-tetramethylpyrazine)是在 1957年首先从 Ligusticim ^ i i中被分离出来的。 四甲基吡嗪具有可可、 咖啡、 乳糖、 洋酒、 格蓬等香气， 广 泛用于食品、 卷烟、 酒类等香晶香料行业。 四甲基吡嗪还是活血行气祛瘀中药川芎中起主 要作用的一种活性生物碱。 近些年来随着对其研究的深入， 人们不断发现其在药学方面的 宝贵价值， 现已作为循环、 中枢神经、 呼吸及其它系统相关疾病的大宗用药， 广泛应用于 心脑血管疾病、 呼吸系统疾病、 肾小球疾病等的治疗。
四甲基吡嗪的生产方法可分为生物合成法、 直接从植物中提取法和化学合成法三种。 利用生物工程技术， 主要指微生物工程技术， 包括菌种筛选诱变技术和发酵工程技术 等等来生产四甲基吡嗪， 目前尚未见国内研究报道， 而国外研究则较多， 主要集中在法、 日、 德、 美等国。
四甲基吡嗪可由多种糖发酵得到。 生物合成法生产四甲基吡嗪的研究现状介绍如下。
Kosuge等人最早证明了微生物能合成吡嗪类物质， 他们发现枯草芽孢杆菌 Bacillus subtilis)有产四甲基吡嗪的能力（1962)。现在人们知道一些细菌和真菌有合成不同烷基 吡嗪的能力 （Seitz， 1994 ; Gallois, 1984)。 Yamaguchi等人曾经利用这些微生物来进行 液体发酵， 但结果发现发酵液中吡嗪浓度太低， 未能达到工业化的要求 （1993)。
Itohiki-natto由于具有独特的吡嗪风味而成为最流行的发酵食品之一。 它是日本人 利用一种被称为纳豆芽孢杆菌 Bacillus natto) 的微生物在大豆固体培养基上发酵而成。 然而， 由于固体发酵所使用的主要是一些经验性的方法， 发酵的结果并不太如人意， 所得 到的香料化合物产量湿重不超过 22微克 /千克 （Kosuge等， 1971 )。
法国 Besscm等人在此领域做了大量工作， 他们利用一株枯草芽孢杆菌 Bacillus subtilis IFO 3013 ) 在大豆固体培养基上进行发酵实验， 得到了 0. 58克 /千克的产量 ( 1998)。这是迄今为止所有文献报道的生物合成法生产四甲基吡嗪的最高产量，但仍尚需 大量的工作才可能工业化生产。
从植物中萃取四甲基吡嗪的方法因原料来源有限， 且原料中四甲基吡嗪的含量太低， 提取成本太高， 无法实现工业化； 化学合成法普遍存在较严峻的环保问题， 反应条件一般 较剧烈， 对设备要求较高， 且产品不是天然的； 生物合成法具有产品绿色天然、'成本低、 反应条件温和、 环境污染小等等突出优点， 是将来四甲基吡嗪合成研究的方向， 有极好的 发展前景， 但从自然界中筛选四甲基吡嗪产生菌是一项十分艰巨的工作， 筛选到的野生菌 确 认 本 的产量又往往非常低， 利用其它微生物技术的起点很低， 所以目前国际上用生物合成法生 产四甲基吡嗪的技术尚未成熟， 未能进行产业化生产。 发明内容
本发明的目的在于针对现有生产四甲基吡嗪的技术难点， 提供一株新分离的以葡萄糖 为底物高产四甲基吡嗪的短小芽孢杆菌， 可以用此菌株发酵生产四甲基吡嗪。
本发明涉及的短小芽孢杆菌名为 Bacillus pumilus RX3-17, 已于 2006年 4月 19日 保藏于中国典型培养物保藏中心， 其保藏编号为 CCTCC M 206043 ο
上述 Bacillus pumilus RX3-17 CCTCC M 206043菌株的生理生化特征如表 1所示。 表 1 Bacillus pumilus RX3-17 CCTCC M 206043菌株的生理生化特征
特征 结果
棒杆状 +
直径 0. 6〜0. 7 μ m 长 1. 5〜3· 0 μ πι 芽孢
椭圆体
肿胀孢子囊
VP反应
VP反应的 pH 利用糖产酸
葡萄糖
阿拉伯糖
木糖
甘露醇
果糖 利用葡萄糖产气 水解反应
酪蛋白 +
明胶 + 淀粉 - 吐温 80 +
七叶灵 - 利用柠檬酸盐 + 利用丙酸盐 - 降解酪氨酸 -
. 苯丙氨酸脱氨酶 -
N03到 N02 - 吲哚 - H 5. 7时生长 + 不同 NaCl浓度时生长
2% NaCl +
5% NaCl +
7% NaCl +
10% NaCl + 不同温度时生长
45。C +
50V +
55 V -
0. 001%溶菌酶时生长 + 精氨酸脱氢酶 一 s pumilus RX3-17 CCTCC M 206043菌株的 16S rDNA序列与其它多株短 小芽孢杆菌的 16S rDNA序列有 99%的相似性。
± Bacillus pumilus RX3-17 CCTCC M 206043菌株， 用于以 SV发酵培养基发酵生 产四甲基吡嗪。 所述 SV发酵培养基配方为： 1升蒸馏水中含： 200克葡萄糖， 50克大豆蛋 白胨， 30克 (NH4) 2HP04， 15毫升的维生素混合液。其中维生素混合液组成为： 1升蒸镏水中 含： 1. 0毫克生物素， 1. 0毫克维生素 B1 5. 0毫克维生素 B2， 25毫克维生素 Ββ， 50毫克 对氨基苯甲酸， 100毫克泛酸钙， 100毫克烟酸。
ϋ Bacillus pumilus RX3-17 CCTCC M 206043菌株， 用于以 SV发酵培养基发酵生 产四甲基吡嗪时， 发酵温度为 37°C， 在 500毫升的锥形瓶中装 50毫升发酵液， 置于摇床 上以 120转 /分钟的转速培养 96〜120小时， 可得到成熟的四甲基吡嗪发酵液。
本发明提供的高产四甲基吡嗪的短小芽孢杆菌解决了微生物发酵法生产四甲基吡嗪中 菌种发酵产物浓度低的瓶颈问题。 微生物发酵法生产四甲基吡嗪具有原料价廉易得、 反应 条件温和、 产品绿色天然、 成本较低、 环境友好等优点。 附图说明
本发明涉及的短小芽孢杆菌名为 Bacillus pumilus RX3-17, 已于 2006年 4月 19日 保藏于中国典型培养物保藏中心， 其保藏编号为 CCTCC M 206043。 附图 1为本发明的 Bacillus pumilus RX3-17 CCTCC M 206043菌株放大 2700倍的电 镜照片。， . 具体实施方式
实施例一：
步骤 1 : Bacillus pumilus RX3-17 CCTCC M 206043菌株的诱变筛选：
从苹果园、 葡萄园等地取得土样， 用生理盐水悬浮后在 80Ό条件下加热 10分钟， 然 后划线涂布到营养琼脂培养皿中， 在 37°C条件下培养。 营养琼脂培养基配方为： 1升蒸馏 水中含： 蛋白胨 10克， 牛肉膏 3克， 氯化钠 5克， 琼脂 18克。 培养基的初始 pH值为 7. 2， 灭菌温度为 121 Ό , 灭菌时间为 15分钟。
挑出单菌落后， 接种于营养肉汤培养基中， 在 37 C条件下置于 120转 /分钟的摇床上 培养。待长至对数生长中期后，将菌体悬浮于 0. 05摩尔 /升的 Tris-马来酸缓冲液（pH 6. 0) 中， 用 0. 5毫克 /升的亚硝基胍处理 30分钟。 釆用稀释法结束诱变过程后， 将诱变培养物 置于摇床上培养 5小时， 然后适当稀释并涂布于营养琼脂培养皿中培养， 用做进一步的筛 选。 营养肉汤培养基制备方法与上述营养琼脂培养基的制备方法相同， 只是不加入琼脂。
由生理生化鉴定实验表明，通过上述实验得到的菌株形态为棒杆状，长 1. 5〜3. 0微米， 直径 0. 6〜0. 7微米， 菌落颜色为乳白色， 产芽孢， VP反应呈阳性， 可利用葡萄糖、 阿拉 伯糖、 木糖、 甘露醇、 果糖产酸， 可水解酪蛋白、 明胶、 吐温 80， 可利用柠檬酸盐， 可在 质量百分比为 10%的 NaCl中生长， 可在 50°C下生长， 其 16S rDNA序列与其它多株短小芽 孢杆菌的 16S rDNA序列有 99%的相似性。
该菌株定名为 Bacillus pumilus RX3-17, 已于 2006年 4月 19日保藏于中国典型培 养物保藏中心， 其保藏编号为 CCTCC M 206043 ο
步骤 2: 制备上述 Bacillus pumilus RX3-17 CCTCC M 206043菌株的细胞液体培养物： 挑取用营养琼脂培养基斜面培养的
Figure imgf000007_0001
pumilus RX3-17 CCTCC M 206043菌株一 环， 接种在装有已灭菌的 25毫升营养肉汤培养基的 300毫升锥形瓶中， 于 37aC、 120转 / 分钟在摇床上培养 12小时， 即制得 ciL pumilus RX3-17 CCTCC M 206043菌株的细 胞液体培养物。
步骤 3： 利用上述 Bacillus pumilus RX3-17 CCTCC M 206043菌株， 以含 160克 /升 葡萄糖的 SV发酵培养基获得成熟的四甲基吡嗪发酵液：
将通过实施例 2的方法获得的 Bacillus pumilus RX3-17 CCTCC M 206043菌株的细胞 液体培养物以体积百分比为 6%的接种量接种到装有已灭菌的 50毫升 SV发酵培养基的 500 毫升锥形瓶中， 于 37Ό、 120转 /分钟在摇床上培养。 在第 96小时四甲基吡嗪的浓度达到 1. 50± 0. 07克 /升， 即得成熟的四甲基吡嗪发酵液。
上述含 160克 /升葡萄糖的 SV发酵培养基配方为： 1升蒸馏水中含： 160克葡萄糖， 50 克大豆蛋白胨， 30克 (NH4) 2HP04， 15毫升维生素混合液， 调节 ρΗ至 7, 0。 其中维生素混合 液组成为： 1升蒸馏水中含： 1. 0毫克生物素， 1. 0毫克维生素 ^， 5. 0毫克维生素 Β2， 25 毫克维生素 Β6， 50毫克对氨基苯甲酸， 100毫克泛酸钙， 100毫克烟酸。 实施例二:利用上述实施例一步骤 1和 2的方法获得的 Bacillus pumilus RX3-17 CCTCC M 206043菌株， 以含 180克 /升葡萄糖的 SV发酵培养基获得成熟的四甲基吡嗪发酵液： 与上述实施例一的步骤 3相同， 所不同的只是将葡萄糖浓度改为 180克 /升。 在第 104 小时得到成熟的发酵液， 其中四甲基吡嗪的浓度达到 1. 79±0. 08克 /升。 实施例三：利用上述实施例一步骤 1和 2的方法获得的 Bacillus pumilus RX3-17 CCTCC 206043菌株， 以含 200克 /升葡萄糖的 SV发酵培养基获得成熟的四甲基吡嗪发酵液： 与实施例一的步骤 3相同， 所不同的只是将葡萄糖浓度改为 200克 /升。在第 112小时 得到成熟的发酵液， 其中四甲基吡嗪的浓度达到 2. 22 ± 0. 06克 /升。 实施例四： 利用上述实施例一步骤 1和 2的方法获得的上述 Bacillus pimilus Wi- VI CCTCC M 206043菌株， 以含 220克 /升葡萄糖的 SV发酵培养基获得成熟的四甲基吡嗪发酵 液：
与实施例一的步骤 3相同， 所不同的只是将葡萄糖浓度改为 220克 /升。在第 120小时 得到成熟的发酵液， 其中四甲基吡嗪的浓度达到 1. 67± 0. 05克 /升。

Claims

权 利 要 求 书
1. 一株高产四甲基吡嗪的短小芽孢杆菌，其特征在于：该短小芽孢杆菌名为 Bacillus pumilus RX3-17, 已于 2006年 4月 19日保藏于中国典型培养物保藏中心， 其保藏编号为 CCTCC M 206043,该 Bacillus pumilus RX3- 17 CCTCC M 206043菌株的细菌学特征是： 形 态为棒杆状， 长 1. 5〜3. 0微米， 直径 0. 6〜0. 7微米， 菌落颜色为乳白色， 产芽孢， VP反 应呈阳性， 可利用葡萄糖、 阿拉伯糖、 木糖、 甘露醇、 果糖产酸， 可水解酪蛋白、 明胶、 吐温 80, 可利用柠檬酸盐， 可在质量百分比为 10%的 NaCl中生长， 可在 50Ό下生长， 所 述 Bacillus pumilus RX3-17 CCTCC M 206043菌株的 16S rDNA序列与其它多株短小芽孢 杆菌的 16S rDNA序列有 99%的相似性。
2. 如权利要求 1所述的高产四甲基吡嗪的短小芽孢杆菌，其特征在于：所逸 Bacillus pumilus RX3-17 CCTCC M 206043菌株， 用于发酵法生产四甲基吡嗪。
3. 如权利要求 2所述的高产四甲基吡嗪的短小芽孢杆菌，其特征在于：所述 BaciUus pumilus RX3-17 CCTCC M 206043菌株用于发酵法生产四甲基吡嗪以 SV发酵培养基发酵生 产四甲基吡嗪。
4. 如权利要求 3 所述的高产四甲基吡嗪的短小芽孢杆菌， 其特征在于： 用所述 Bacillus pumilus RX3-17 CCTCC M 206043菌株以 SV发酵培养基发酵生产四甲基吡嗪， 所述 SV发酵培养基配方为： 1升蒸馏水中含： 160〜220克葡萄糖， 50克大豆蛋白胨， 30 克 (NH4) 2HP04， 15毫升维生素混合液。
5. 如权利要求 4所述的高产四甲基吡嗪的短小芽孢杆菌， 其特征在于： 所述 SV发酵 培养基配方中维生素混合液组成为'： 1升蒸馏水中含： 1. 0毫克生物素， 1. 0毫克维生素 B,， 5. 0毫克维生素 B2， 25毫克维生素 ' Ββ， 50毫克对氨基苯甲酸， 100毫克泛酸钙， 100毫克 烟酸。
6. 如权利要求 2或 3所述的高产四甲基吡嗪的短小芽孢杆菌， 其特征在于： 用所述 Bacillus pumilus RX3-17 CCTCC M 206043菌株以 SV发酵培养基发酵生产四甲基吡嗪， 发酵温度为 37°C， 在 500毫升的锥形瓶中装 50毫升发酵液， 置于摇床上以 120转 /分钟的 转速培养 96〜120小时， 可得到成熟的四甲基吡嗪发酵
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