一株费氏丙酸杆菌生长特性及其代谢物抑菌作用分析

对一株费氏丙酸杆菌生长特性及其代谢物抑菌作用进行研究。结果表明：该菌株生长周期约为10 d,第1～3天为其对数生长期,第4～8天为菌体生长的稳定期,第9～10天为衰亡期。在生长过程中,菌体利用糖的速率很快,还原糖在发酵1 d内几乎被消耗殆尽,发酵液pH值在发酵的第1天下降到4.6左右,此后一直到第10天,pH值基本稳定在4.6左右。在同等条件下,该丙酸杆菌代谢物对大肠杆菌的抑制作用优于对金黄色葡萄球菌和酵母菌的抑制作用。对代谢产物的分析结果表明,发酵产物中抑菌物质除了丙酸之外,还存在其他物质,初步判定为细菌素。     

Evaluation of in vitro antimicrobial activity of Thai basil oils and their micro-emulsion formulas against Propionibacterium acnes

The aim of this study was to evaluate the efficacy of Thai basil oils and their micro-emulsions, on in vitro activity against Propionibacterium acnes. An agar disc diffusion method was employed for screening antimicrobial activity of the essential oils of Ocimum basilicum L. (sweet basil), Ocimum sanctum L. (holy basil) and Ocimum americanum L. (hoary basil) against P. acnes. Minimum inhibitory concentration (MIC) values of the basil oils were determined using an agar dilution assay. The obtained results indicated that the MIC values of sweet basil and holy basil oils were 2.0% and 3.0% v/v, respectively, whereas hoary basil oil did not show activity against P. acnes at the highest concentration tested (5.0% v/v). Gas chromatography-mass spectrometry analysis revealed that methyl chavicol (93.0%) was the major compound in sweet basil oil, and eugenol (41.5%), gamma-caryophyllene (23.7%) and methyl eugenol (11.8%) were major compounds in holy basil oil. Hoary basil oil contained high amounts of geraniol (32.0%) and neral (27.2%) and small amounts of methyl chavicol (0.8%). The Oil-in-water (o/w) micro-emulsions of individual basil oils with concentrations corresponding to their MIC values were formulated. The stable o/w micro-emulsion system for basil oil consisted of 55.0% v/v water phase, 10.0% v/v oil phase (2.0 or 3.0% v/v sweet basil or 3.0% v/v holy basil oil plus 7.0% v/v isopropyl myristate), 29.2% v/v polysorbate 80 and 5.8% v/v 1,2-propylene glycol. Hydroxyethylcellulose at a concentration of 0.5% w/v was used as thickening agent. According to the disc diffusion assay, the formulations containing sweet basil oil exhibited higher activity against P. acnes than those containing holy basil oil, and the thickened formulations tended to give a lower activity against P. acnes than the non-thickened formulations. The prepared micro-emulsions were stable after being tested by a heat-cool cycling method for five cycles. These findings indicate the possibility to use Thai sweet and holy basil oil in suitable formulations for acne skin care.     

Capsular exopolysaccharide biosynthesis gene of Propionibacterium freudenreichii subsp. shermanii

In the dairy industry, exopolysaccharides (EPS) contribute to improving the texture and viscosity of cheese and yoghurt and also receive increasing attention because of their beneficial properties for health. For lactic acid bacteria, the production of EPS is well studied. However, for dairy propionibacteria the biosynthesis of EPS is poorly documented. A polysaccharide synthase-encoding gene was identified in the genome of Propionibacterium freudenreichii subsp. shermanii TL 34 (CIP 103027). This gene best aligns with Tts, the polysaccharide synthase gene of Streptococcus pneumoniae type 37 that is responsible for the production of a beta-glucan capsular polysaccharide. PCR amplification showed the presence of an internal fragment of this gene in twelve strains of P. freudenreichii subsp. shermanii with a ropy phenotype in YEL+ medium. The gene sequence is highly conserved, as less than 1% of nucleotides differed among the 10 strains containing the complete gtf gene. The same primers failed to detect the gene in Propionibacterium acidipropionici strain TL 47, which is known to excrete exopolysaccharides in milk. The presence of (1-->3, 1-->2)-beta-d-glucan capsule was demonstrated for 7 out of 12 strains by agglutination with a S. pneumoniae-type 37-specific antiserum. The presence of mRNA corresponding to the gene was detected by RT-PCR in three strains at both exponential and stationary growth phases. This work represents the first identification of a polysaccharide synthase gene of P. freudenreichii, and further studies will be undertaken to elucidate the role of capsular EPS.     

胸膜肺炎放线杆菌血清型特异的疫苗候选基因的筛选及痤疮丙酸杆菌异源免疫

目的筛选猪胸膜肺炎放线杆菌(APP)1型与5型特异的疫苗候选基因,并研究痤疮丙酸杆菌对猪传染性胸膜肺炎的异源免疫。方法采用cDNA代表性差异分析、大肠杆菌核糖体展示、免疫筛选和RT-PCR技术筛选APP1型和5型血清型特异的疫苗候选基因,进行克隆、测序及同源序列分析。并用与APP疫苗候选基因具有同源序列的痤疮丙酸杆菌(PA)免疫小鼠,研究其对APP感染的异源免疫。结果获得血清1型APP6条疫苗候选基因,2条为未知基因,2条与PA同源性为98%,2条与人cDNA有同源性。获得血清5型APP7条疫苗候选基因,其中2条为未知基因,2条与PA同源性分别为93%和100%。用PA全菌免疫小鼠,产生的抗血清可与1型和5型APP发生强的免疫反应,ELISA检测效价达1∶3200;脾淋巴细胞检测结果显示,免疫组CD3+、CD4+T细胞的百分率及CD4+/CD8+比值与对照组相比均升高,两组CD3+T细胞百分率差异有显著意义。以10倍LD501型和5型APP分别感染PA免疫小鼠,小鼠存活率分别为95%和90%,并且在攻毒后第15天体内APP可全部被清除。结论1型与5型APP存在不同的疫苗候选基因,痤疮丙酸杆菌与之存在共同抗原,能够产生交叉免疫应答,对血清1型和5型APP的感染具有预防作用。     

维生素B_(12)的生物合成研究

维生素B12广泛应用于医药、食品和畜牧业,主要由微生物发酵得到。脱氮假单胞菌(Pseudomonas denitrificans)和费氏丙酸杆菌(Propionibacterium freudenreichii)是主要的生产菌种。与之相应,维生素B12存在好氧和厌氧2条生物合成途径,生物合成过程十分复杂,2条合成途径大体相同又各有特点。维生素B12发酵产量的提高有待于菌种的改良和发酵工艺的改进。了解维生素B12生物合成途径和代谢调控机制具有重要意义,可为育种工作提供理论基础。     

费氏丙酸杆菌生物学特性及与5株益生菌拮抗性试验研究

为了评价费氏丙酸杆菌的益生特性,费氏丙酸杆菌活化后,划线和镜检观察其形态特征,然后利用体外共培养法测定费氏丙酸杆菌对3种致病菌的抑制效果,同时利用平板计数法研究费氏丙酸杆菌的耐酸性、耐胆盐和耐高温等生物学特性,以及5株益生菌拮抗性试验。结果表明,费氏丙酸杆菌对大肠杆菌和沙门氏菌没有显著的抑制作用,而对金黄色葡萄球菌的抑制效果明显（P＜0.01）;其在pH值为2.0条件下维持3 h,活菌数降低一个数量级;在胆盐含量为0.30%条件下维持6 h,存活率为18.67%;在60 ℃下处理20 min,存活率为12%,因此费氏丙酸杆菌具有一定的耐酸、耐高温能力以及很强的耐胆盐活性。除枯草芽孢杆菌与丁酸梭菌有拮抗作用外,其余菌株间均没有拮抗作用。     

鲜牛奶中丙酸杆菌的筛选、鉴定及特性分析

从新鲜生牛奶中分离筛选产丙酸较高的菌株,经形态学特征、生理生化及糖发酵试验、16S rDNA基因序列同源性分析以及多位点序列分型(multilocus sequence typing)等实验鉴定该菌株,分析了其对不同碳源的利用率及产酸情况,并从溶血性试验及抗生素抗性试验方面来评估该菌株的安全性。结果表明,从4批次样品中共分离获得54株能产丙酸的菌株,其中一株菌的丙酸产量达到7.38 g/L,为费氏丙酸杆菌(Propionibacterium freudenreichii)。比较了葡萄糖和甘油对其生长的影响,发现其对葡萄糖的利用率大于对甘油的利用率,在含葡萄糖的培养基中于30 ℃厌氧培养120 h后丙酸产量达到7.38 g/L。该菌株无溶血性,对卡那霉素等氨基糖苷类抗生素有耐药性,对氨苄西林、万古霉素、氯霉素、克林霉素、四环素敏感,对红霉素中介。综上,费氏丙酸杆菌B1具有一定的潜在应用价值。     

Aerobic culture of Propionibacterium freudenreichii ET-3 can increase production ratio of 1,4-dihydroxy-2-naphthoic acid to menaquinone

This is the first report on the production of both 1,4-dihydroxy-2-naphthoic acid (DHNA) and menaquinone by Propionibacterium freudenreichii ET-3. DHNA can be a stimulator of bifidogenic growth, and menaquinone has important roles in blood coagulation and bone metabolism. During anaerobic culture, DHNA and menaquinone concentrations reached 0.18 mM and 0.12 mM, respectively. The molar ratio between these products was approximately 3:2, which was not affected by culture pH and temperature over the ranges of 6.0-7.0 and 31-35 degrees C, respectively. As for organic acid, propionate and acetate accumulated at concentrations of 0.3 M and 0.15 M, respectively, and the propionate accumulation particularly inhibited further production of DHNA. To improve DHNA production, we switched from anaerobic condition to aerobic condition during the culture when lactose was depleted. DHNA concentration continued to increase even after lactose exhaustion, reaching 0.24 mM. In contrast to DHNA production, menaquinone production stopped after the switch to aerobic condition. The total molar production of DHNA and menaquinone was 0.3 mM irrespective of aerobic culture and anaerobic-aerobic switching culture. Therefore, the anaerobic-aerobic switching culture could increase the production ratio of DHNA to menaquinone. The DHNA concentration obtained from the anaerobic-aerobic switching culture was 1.3-fold higher than that in the anaerobic culture, because P. freudenreichii ET-3 utilized propionate accumulated in the medium via the reversed methylmalonyl CoA pathway under aerobic condition. The culture method proposed in this study could be applicable to industrial-scale fermentation using 1000 l of media, by which 0.23 mM DHNA was produced.     

在内置式植物纤维床反应器中用糖蜜固定化发酵制丙酸

开发了一种综合利用制糖工业副产物生产丙酸的新方法,将制糖工业副产物甘蔗渣和糖蜜分别作为固定化载体和碳源,应用于丙酸生产.以甘蔗渣为固定化载体构建了一种内置式植物纤维床反应器(Inner plant fibrous-bed bioreactor,IPFB).以Propionibacterium freudenreichii CCTCC M207015为发酵菌株,对在IPFB中分别用未处理糖蜜及糖蜜水解液为碳原发酵制丙酸进行考察.以未处理糖蜜为碳源发酵,254 h丙酸浓度为52.39 g·L~(-1),丙酸生产速率为0.21 g·(L·h)~(-1);糖蜜水解液为碳源发酵,254 h后丙酸浓度达75.18 g·L~(-1),丙酸生产速率达0.30 g·(L·h)~(-1),丙酸占总有机酸比例达83.10%(w/w).稳定性实验表明以糖蜜水解液为碳源,利用IPFB发酵生产丙酸10批仍然保持较高的丙酸生产效率.扫描电镜显示大量丙酸杆菌的细胞吸附于甘蔗渣表面与间隙中,有效实现了高密度发酵.     

不同初始pH对丙酸杆菌细菌素发酵的影响

薛氏丙酸杆菌在不同初始pH下摇瓶厌氧发酵,测定发酵液的pH、生物量、还原糖、氨基氮及抑菌活性,探求不同初始pH对丙酸杆菌细菌素发酵的影响。将初始pH为6.0,6.5,7.0,7.5,8.0的培养基分批进行发酵,结果显示:丙酸杆菌在初始pH为6.5时,发酵第2d的生物量为8.4mg/mL,与其他初始pH的差异极显著(P<0.01);以酵母菌和恶臭假单胞菌为指示菌,发酵产物细菌素的抑菌活性在第7d达到最大,初始pH6.5的抑菌活性分别为19.203AU/mL和24.827AU/mL,与其他初始pH的差异均极显著(P<0.01),说明不同初始pH对丙酸杆菌细菌素发酵有很大的影响,其最适初始pH为6.5。