BioVector? 丁酸梭菌 ATCC 19398 標(biāo)準(zhǔn)菌株

BioVector? Clostridium butyricum ATCC 19398? / Type Strain

第一部分：中文說明

一、 產(chǎn)品基本信息與分類學(xué)背景

• 菌種名稱：丁酸梭菌（Clostridium butyricum），又稱酪酸菌。

• 菌株編號(hào)：ATCC 19398

• 同義菌株別名：NCTC 7423、DSM 10702、JCM 1391、IFO 13949、VPI 3266。

• 分類學(xué)地位：厚壁菌門（Bacillota）、梭菌綱（Clostridia）、梭菌目（Eubacteriales）、梭菌科（Clostridiaceae）、梭菌屬（Clostridium）。

• 模式菌株（Type Strain）：是（Yes）。該菌株為丁酸梭菌的國際官方標(biāo)準(zhǔn)模式物種鑒定基準(zhǔn)株。

• 物種分離來源：最初分離自豬的腸道（Intestine of pig）。

• 生物安全級(jí)別：1級(jí)（BSL-1）。該菌株為無致病性的益生性細(xì)菌，不產(chǎn)生肉毒毒素（Type E botulinum toxin）等梭菌屬常見毒素，屬于食品及飼料級(jí)安全菌株。

二、 細(xì)菌形態(tài)學(xué)、基因組與生理生化特征

• 細(xì)胞形態(tài)：革蘭氏染色陽性（Gram-positive），呈現(xiàn)直或微彎的桿狀，細(xì)胞大小通常為 $1.0\text{--}1.6\ \mu m \times 3.0\text{--}8.0\ \mu m$。在環(huán)境不良時(shí)，菌體可形成卵圓性的次極端或極端芽孢（Subterminal / Terminal Endospore），導(dǎo)致菌體膨大呈梭狀。

• 基因組信息：

  • 組裝大小：全基因組大小約為 4.68 Mb（單染色體，部分含有質(zhì)粒）。

  • GC 含量：28.73%。

• 代謝特征：

  • 屬于嚴(yán)格厭氧菌（Strict Anaerobe）。

  • 具備極強(qiáng)的專性發(fā)酵能力，能利用多種碳水化合物（如葡萄糖、麥芽糖、蔗糖）作為底物，代謝的核心終產(chǎn)物為大量的丁酸（Butyric Acid，即酪酸）、醋酸和二氧化碳（$CO_2$）及氫氣（$H_2$）。

  • 細(xì)胞內(nèi)可聚積形似支鏈淀粉的 $\alpha$-聚糖（Granulose）作為儲(chǔ)備碳源。

三、 培養(yǎng)基配方與理化參數(shù)

• 推薦標(biāo)準(zhǔn)培養(yǎng)基：

  • ATCC Medium 2107（Modified Reinforced Clostridial Medium，增強(qiáng)型梭菌培養(yǎng)基）

  • PYG Medium（Peptone-Yeast-Glucose Broth，蛋白胨-酵母膏-葡萄糖培養(yǎng)基）

  • Brucella Blood Agar（布氏血瓊脂平板）：適用于菌落純度分析及形態(tài)學(xué)觀察。

• 物理培養(yǎng)參數(shù)：

  • 培養(yǎng)溫度：37°C 恒溫培養(yǎng)。

  • 氣體環(huán)境：嚴(yán)格厭氧（100% $N_2$ 或厭氧產(chǎn)氣包、厭氧工作站）。

  • 狀態(tài)指示：若培養(yǎng)基中包含氧化還原指示劑高Resazurin（刃天青），使用前或培養(yǎng)期間若液體變?yōu)?strong data-path-to-node="9,1,1,2,0" data-index-in-node="48" style="line-height:1.15 !important;margin-top:0px !important;">粉紅色，說明氧氣逸入已被氧化，必須予以棄用。

四、 菌株復(fù)蘇、擴(kuò)增與保存標(biāo)準(zhǔn)步驟

1. 凍干粉或冷凍管復(fù)蘇：

   • 準(zhǔn)備經(jīng)嚴(yán)格預(yù)還原（Pre-reduced）脫氧處理的 #2107 或 PYG 液體培養(yǎng)基 5–6 mL。

   • 在厭氧操作臺(tái)內(nèi)（或通過厭氧套管氣流保護(hù)）打開安瓿瓶，吸取 0.5 mL 液體培養(yǎng)基將菌塊完全重懸，隨后轉(zhuǎn)入主培養(yǎng)管中。

   • 可同步蘸取少量懸液接種于厭氧血瓊脂平板上，以觀察單菌落形態(tài)。

2. 常規(guī)孵育與傳代：

   • 將接種好的試管（擰松管蓋）置于厭氧培養(yǎng)箱或密閉厭氧罐（放入活性厭氧產(chǎn)氣包）中，37°C 培養(yǎng) 24–48 小時(shí)。

   • 控制對(duì)照：同步將一塊接種板置于普通有氧培養(yǎng)箱中進(jìn)行有氧培養(yǎng)，用以檢測操作過程中是否混入了好氧性雜菌（正常情況下有氧板應(yīng)無任何菌落生長）。

3. 長期菌種保存：

   • 待液體培養(yǎng)物生長至對(duì)數(shù)末期，按 1:1 的體積比混入無菌的 30% 甘油保護(hù)劑（最終甘油濃度為 15%）。

   • 分裝于耐冷凍管中，迅速移入 -80°C 超低溫冰箱或液氮中長期凍存。由于該菌易形成抗逆性極強(qiáng)的芽孢，亦可通過真空干燥法制備凍干粉保存。

五、 核心科研與工業(yè)應(yīng)用方向

1. 新型益生菌與腸道微生態(tài)研究：作為丁酸梭菌的國際標(biāo)準(zhǔn)型菌株，它被廣泛用作對(duì)照組，用于評(píng)估各類野生分離株在調(diào)節(jié)宿主腸道菌群、對(duì)抗艱難梭菌（Clostridioides difficile）感染以及分泌短鏈脂肪酸（SCFAs）方面的生物學(xué)效應(yīng)。

2. 生物能源與化學(xué)品發(fā)酵（丁酸與氫氣轉(zhuǎn)化）：利用其高效率的厭氧發(fā)酵特性，作為工程指示菌開展代謝工程研究，優(yōu)化從木質(zhì)纖維素或生物質(zhì)低成本基質(zhì)向丁酸、有機(jī)溶劑、以及清潔能源氫氣（$H_2$）的工業(yè)轉(zhuǎn)化工藝。

3. 食品、奶酪工業(yè)品質(zhì)控制（抗性評(píng)價(jià)）：在乳制品 and 奶酪工業(yè)中，梭菌屬常導(dǎo)致奶酪發(fā)生“遲發(fā)性產(chǎn)氣”（Late Blowing Defect）變質(zhì)。該標(biāo)準(zhǔn)株常被用于食品防腐劑效能評(píng)估以及新型巴氏殺菌、高壓滅菌工藝對(duì)梭菌芽孢殺滅率的標(biāo)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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