BioVector® 丁酸梭菌 ATCC 19398 标准菌株

BioVector® Clostridium butyricum ATCC 19398™ / Type Strain

第一部分：中文说明

一、 产品基本信息与分类学背景

• 菌种名称：丁酸梭菌（Clostridium butyricum），又称酪酸菌。

• 菌株编号：ATCC 19398

• 同义菌株别名：NCTC 7423、DSM 10702、JCM 1391、IFO 13949、VPI 3266。

• 分类学地位：厚壁菌门（Bacillota）、梭菌纲（Clostridia）、梭菌目（Eubacteriales）、梭菌科（Clostridiaceae）、梭菌属（Clostridium）。

• 模式菌株（Type Strain）：是（Yes）。该菌株为丁酸梭菌的国际官方标准模式物种鉴定基准株。

• 物种分离来源：最初分离自猪的肠道（Intestine of pig）。

• 生物安全级别：1级（BSL-1）。该菌株为无致病性的益生性细菌，不产生肉毒毒素（Type E botulinum toxin）等梭菌属常见毒素，属于食品及饲料级安全菌株。

二、 细菌形态学、基因组与生理生化特征

• 细胞形态：革兰氏染色阳性（Gram-positive），呈现直或微弯的杆状，细胞大小通常为 $1.0\text{--}1.6\ \mu m \times 3.0\text{--}8.0\ \mu m$。在环境不良时，菌体可形成卵圆性的次极端或极端芽孢（Subterminal / Terminal Endospore），导致菌体膨大呈梭状。

• 基因组信息：

  • 组装大小：全基因组大小约为 4.68 Mb（单染色体，部分含有质粒）。

  • GC 含量：28.73%。

• 代谢特征：

  • 属于严格厌氧菌（Strict Anaerobe）。

  • 具备极强的专性发酵能力，能利用多种碳水化合物（如葡萄糖、麦芽糖、蔗糖）作为底物，代谢的核心终产物为大量的丁酸（Butyric Acid，即酪酸）、醋酸和二氧化碳（$CO_2$）及氢气（$H_2$）。

  • 细胞内可聚积形似支链淀粉的 $\alpha$-聚糖（Granulose）作为储备碳源。

三、 培养基配方与理化参数

• 推荐标准培养基：

  • ATCC Medium 2107（Modified Reinforced Clostridial Medium，增强型梭菌培养基）

  • PYG Medium（Peptone-Yeast-Glucose Broth，蛋白胨-酵母膏-葡萄糖培养基）

  • Brucella Blood Agar（布氏血琼脂平板）：适用于菌落纯度分析及形态学观察。

• 物理培养参数：

  • 培养温度：37°C 恒温培养。

  • 气体环境：严格厌氧（100% $N_2$ 或厌氧产气包、厌氧工作站）。

  • 状态指示：若培养基中包含氧化还原指示剂高Resazurin（刃天青），使用前或培养期间若液体变为粉红色，说明氧气逸入已被氧化，必须予以弃用。

四、 菌株复苏、扩增与保存标准步骤

1. 冻干粉或冷冻管复苏：

   • 准备经严格预还原（Pre-reduced）脱氧处理的 #2107 或 PYG 液体培养基 5–6 mL。

   • 在厌氧操作台内（或通过厌氧套管气流保护）打开安瓿瓶，吸取 0.5 mL 液体培养基将菌块完全重悬，随后转入主培养管中。

   • 可同步蘸取少量悬液接种于厌氧血琼脂平板上，以观察单菌落形态。

2. 常规孵育与传代：

   • 将接种好的试管（拧松管盖）置于厌氧培养箱或密闭厌氧罐（放入活性厌氧产气包）中，37°C 培养 24–48 小时。

   • 控制对照：同步将一块接种板置于普通有氧培养箱中进行有氧培养，用以检测操作过程中是否混入了好氧性杂菌（正常情况下有氧板应无任何菌落生长）。

3. 长期菌种保存：

   • 待液体培养物生长至对数末期，按 1:1 的体积比混入无菌的 30% 甘油保护剂（最终甘油浓度为 15%）。

   • 分装于耐冷冻管中，迅速移入 -80°C 超低温冰箱或液氮中长期冻存。由于该菌易形成抗逆性极强的芽孢，亦可通过真空干燥法制备冻干粉保存。

五、 核心科研与工业应用方向

1. 新型益生菌与肠道微生态研究：作为丁酸梭菌的国际标准型菌株，它被广泛用作对照组，用于评估各类野生分离株在调节宿主肠道菌群、对抗艰难梭菌（Clostridioides difficile）感染以及分泌短链脂肪酸（SCFAs）方面的生物学效应。

2. 生物能源与化学品发酵（丁酸与氢气转化）：利用其高效率的厌氧发酵特性，作为工程指示菌开展代谢工程研究，优化从木质纤维素或生物质低成本基质向丁酸、有机溶剂、以及清洁能源氢气（$H_2$）的工业转化工艺。

3. 食品、奶酪工业品质控制（抗性评价）：在乳制品 and 奶酪工业中，梭菌属常导致奶酪发生“迟发性产气”（Late Blowing Defect）变质。该标准株常被用于食品防腐剂效能评估以及新型巴氏杀菌、高压灭菌工艺对梭菌芽孢杀灭率的标定。

PART 2: ENGLISH SECTION

I. General Information and Taxonomic Background

• Organism Name: Clostridium butyricum

• Strain Identification: ATCC 19398

• Alternative Strain Aliases: NCTC 7423, DSM 10702, JCM 1391, IFO 13949, VPI 3266.

• Taxonomic Lineage: Phylum Bacillota; Class Clostridia; Order Eubacteriales; Family Clostridiaceae; Genus Clostridium.

• Type Strain Designation: Yes. This culture represents the international official standard reference type strain used for species-level verification and comparative studies.

• Isolation Source: Originally isolated from the intestine of a pig.

• Biosafety Level: BSL-1. Non-pathogenic, non-zoonotic probiotic agent. It lacks any genes for botulinum neurotoxins or other severe clostridial enteropathogens, ensuring qualified compliance for general laboratory maintenance.

II. Morphological, Genomic, and Physiological Attributes

• Cell Morphology: Gram-positive, straight or slightly curved rod-shaped bacilli measuring approximately $1.0\text{--}1.6\ \mu m \times 3.0\text{--}8.0\ \mu m$. Under nutrient starvation, it yields oval subterminal or terminal endospores that cause the mother cell to swell into a characteristic spindle form.

• Genomic Profile:

  • Assembly Length: Placed at approximately 4.68 Mb (comprising a circularized main chromosome).

  • GC Content: 28.73%.

• Metabolic Pattern:

  • Classed as a strict (obligate) anaerobe.

  • Ferments an array of carbohydrates (including glucose, maltose, and sucrose), leading to the accumulation of butyric acid as its principal volatile fatty acid byproduct, flanked by acetate, carbon dioxide ($CO_2$), and molecular hydrogen ($H_2$).

  • Accumulates an intracellular amylopectin-like $\alpha$-polyglucan (granulose) matrix as a temporary energy storage reserve.

III. Medium Composition and Physical Cultivation Metrics

• Recommended Cultivation Media:

  • ATCC Medium 2107: Modified Reinforced Clostridial Broth.

  • DSMZ Medium 104 / 110: Peptone-Yeast-Glucose (PYG) Broth.

  • Brucella Blood Agar Plates: Highly recommended for checking colony purity, verifying single-colony morphotypes, and general scaling.

• Physical Environmental Windows:

  • Incubation Temperature: Kept steady at 37°C.

  • Gaseous Headspace: Strictly anaerobic atmosphere (100% $N_2$ gas streams, anaerobic chambers, or validated catalyst-driven gas-pack jars).

  • Oxidation Warning: When utilizing media supplemented with Resazurin indicator, any visible pink discoloration indicates oxygen infiltration and oxidation. Such media must be discarded immediately.

IV. Recovery, Propagation, and Storage Procedures

1. Thawing and Initial Inoculation:

   • Work inside an anaerobic chamber or use a gas cannula system. Use freshly prepared, pre-reduced media to ensure optimal viability.

   • Rehydrate the freeze-dried pellet or thawed suspension by transferring 0.5 mL of pre-reduced #2107 broth into the primary vial, mixing thoroughly, and returning the whole volume back into a 5–6 mL culture tube.

   • Streak a few droplets onto a Brucella Blood Agar plate to verify isolation purity.

2. Incubation and Contamination Controls:

   • Keep tube caps loose and incubate at 37°C for 24–48 hours inside an anaerobic environment.

   • Quality Control Check: Parallelly incubate one inoculated agar plate aerobically at 37°C. The complete absence of aerobic growth validates that the culture has not been cross-contaminated with facultative or obligate aerobes.

3. Cryopreservation Protocols:

   • Harvest active cultures near the late-exponential phase. Add an equal volume of sterile 30% glycerol solution (yielding a 15% final concentration).

   • Aliquot into cryovials and plunge directly into a -80°C ultra-low freezer or liquid nitrogen. Long-term storage via lyophilization is also effective due to its robust endospore survival rates.

V. Core Industrial and Scientific Research Verticals

1. Probiotic Reference Standards & Microbiome Therapeutics: Utilized globally as the definitive baseline type strain to cross-examine wild-type therapeutic isolates regarding their capacity to secrete short-chain fatty acids (SCFAs), repair gut barrier integrity, and antagonize enteropathogens like Clostridioides difficile.

2. Bioenergy & Metabolic Fermentation (Butyrate & Hydrogen Yields): Serves as an ideal platform for metabolic engineering and bioprocess design, focusing on optimizing the continuous conversion of lignocellulosic biomass or low-cost industrial carbohydrates into premium butyric acid and green hydrogen ($H_2$) gas.

3. Dairy Quality Control & Spoilage Assays: Since Clostridium species are notorious for inducing the "late blowing defect" (LBD) in semi-hard and hard cheeses, this standard reference strain is heavily deployed to screen natural antimicrobial preservatives and validate the inactivation curves of pasteurization or ultra-high pressure sterilization methods against bacterial endospores.

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