Improved medium for selective isolation and enumeration of Bifidobacterium.

Petuely's selective medium for Bifidobacterium was improved by addition of riboflavin, nucleic acid bases, pyruvic acid, and nalidixic acid. The modified medium, when examined under strictly anaerobic conditions for efficient isolation of Bifidobacterium from human fecal samples, exhibited selective and high viable counts that were close to those found on the usual nonselective medium.     

Selective medium for isolation and enumeration of Bifidobacterium spp

A new method was developed for the isolation and enumeration of Bifidobacterium spp. from natural aquatic environments. The method was based on the utilization of a new medium, Bifidobacterium iodoacetate medium 25, and resuscitation techniques were used to isolate injured bifidobacteria. The new medium was tested with a nonselective reference medium on sewage and sewage-polluted surface waters. Relatively little colonial growth of any other bacterial genera occurred; when such colonies did grow, Bifidobacterium could be easily differentiated by its colonial morphology or, after Gram staining, by its typical bifidobacterial morphology.     

一株携带质粒的人两歧双歧杆菌的分离与鉴定

目的:分离携带天然质粒的人双歧杆菌.方法:用自制的改良型Blb双歧杆菌选择培养基,从人新鲜粪便分离双歧杆菌,对初步质粒检测阳性的单菌落通过糖发酵试验、(G C)mol%测定和16S rDNA序列分析,进行菌株鉴定.结果:筛选到一株携带天然质粒的人双歧杆菌,编号B200304,在1.0%琼脂糖凝胶上,测得质粒的相对分子质量约为22 kb.通过对该菌株的形态学观察和糖发酵试验等生理生化特征研究,证明该菌株为两歧双歧杆菌(Bifidobacterium bifidum);HPLC法测得其(G C)mol%为55.6,16SrDNA序列分析进一步证实该菌株为两歧双歧杆菌.结论:分离得到一株携带天然质粒的人两歧双歧杆菌新菌株.     

Selective medium for isolation and enumeration of Bifidobacterium spp.

A new method was developed for the isolation and enumeration of Bifidobacterium spp. from natural aquatic environments. The method was based on the utilization of a new medium, Bifidobacterium iodoacetate medium 25, and resuscitation techniques were used to isolate injured bifidobacteria. The new medium was tested with a nonselective reference medium on sewage and sewage-polluted surface waters. Relatively little colonial growth of any other bacterial genera occurred; when such colonies did grow, Bifidobacterium could be easily differentiated by its colonial morphology or, after Gram staining, by its typical bifidobacterial morphology.     

大豆多糖对双歧杆菌及人肠道菌群生长的影响

目的研究大豆多糖对双歧杆菌及肠道菌群生长的影响。方法替换Bs培养基中的碳源,分为不加糖、加葡萄糖2％、加大豆多糖2％、加大豆多糖5％、加低聚果糖2％五组,加3种双歧杆菌（长双歧、青春双歧、两歧双歧）菌液1％,测其24h后的活菌数,比较大豆多糖对双歧杆菌生长的影响;替换Bs培养基中的碳源,分为不加糖、加葡萄糖2％、加大豆多糖2％,加低聚果糖2％四组,加人体粪便菌液1％,模拟人体肠道环境厌氧培养24h后,用选择性培养基测其肠杆菌、肠球菌、双歧杆菌、乳酸杆菌的活菌数,观察大豆多糖对人体肠道菌群的影响。结果大豆多糖添加量为5％时对长双歧的促进作用明显优于不加糖组（P〈0．05）;大豆多糖对人体肠道各菌群的生长促进作用与低聚果糖差异无显著性（P〉0．05）。结论大豆多糖对长双歧杆菌的体外促进作用较明显;以粪菌群发酵糖试验表明,大豆多糖对乳杆菌和双歧杆菌均有促进作用,和低聚果糖作用效果相比差异无显著性（P〉0．05）,具有益生元的特性。     

一株双歧杆菌质粒聚合酶基因的PCR扩增和鉴定

目的PCR扩增人双歧杆菌天然质粒的聚合酶基因。方法用改良型MRS双歧杆菌选择培养基,从人新鲜粪便分离长双歧杆菌,PCR扩增长双歧杆菌质粒聚合酶(Bifidobacterium plasmid polymerase,BPP)基因,对BPP基因检测阳性的PCR产物通过序列分析,进行鉴定。结果人长双歧杆菌天然质粒的聚合酶基因PCR扩增后,经1.0%琼脂糖凝胶电泳,测得BPP基因的相对分子质量约为1.9 kb。通过BLAST序列比对分析与GenBank中相应基因同源性为96%。结论成功克隆了1株双歧杆菌天然质粒的聚合酶基因,为构建与双歧杆菌宿主质粒相适应的载体奠定了基础。     

A New Selective Medium for Bifidobacterium spp.

A new selective antibiotic-free medium for Bifidobacterium spp. is defined. This medium has lactulose as the main carbon source and includes methylene blue, propionic acid, and lithium chloride as inhibitors of some related bacterial species. The low pH of the medium contributes to the inhibition of the growth of Enterobacteriaceae. This new selective medium has a simple composition, and the level of recovery it yields is similar to those yielded by nonselective media for Bifidobacterium strains. It could thus be used for routine analysis in environmental or food microbiology.     

一种改良大便双歧杆菌计数方法

目的：研制一种大便双歧杆菌鉴别计数培养基。方法：利用大多数双歧杆菌具有半乳糖苷酶且活性较高的特性,在自制改良BS培养基中加入X-Gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷）作为底物,双歧杆菌水解底物释放出吲哚,产生颜色反应。结果：双歧杆菌菌落呈深蓝色。乳酸菌和其它细菌一般为白色或淡蓝色。结论：自制改良BS培养基上双歧杆菌与乳酸菌及其他细菌菌落有明显的鉴别性。适用于大便双歧杆菌鉴别计数。     

Bifidobacterium-selective isolation and enumeration from chicken caeca by a modified oligosaccharide antibiotic-selective agar medium

AIMS: To determine the efficacy and selectivity of an acidified, antibiotic-selective, oligosaccharide-containing media for enumerating Bifidobacterium spp. from chicken caeca samples. METHODS AND RESULTS: Transoligosaccharide propionate agar medium (TOS) modified by addition of mupirocin (50 microg ml-1) and glacial acetic acid (1%, v/v), did not inhibit the growth of bifidobacteria compared with the control media yet inhibited the growth of Lactobacillus acidophilus, Lactobacillus gallinarum, Lactobacillus helveticus and Streptococcus gordonii. CONCLUSIONS: Addition of mupirocin (50 microg ml-1) and glacial acetic acid (1%, v/v) to TOS (TOS-AM50), is an effective selective medium for isolation and enumeration of Bifidobacterium spp. from chicken caeca samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of an intestinal bifidobacteria-selective media contributes to the study of probiotics and prebiotics in poultry and potentially other species.     

菊花水煎液对长双歧杆菌促生长作用的探讨

目的研究菊花水煎液促进长双歧杆菌的体外增殖与活性的作用,初步探讨它作为一种双歧因子的条件。方法以菌液A600值、pH、活菌计数及发酵牛乳的凝乳时间、pH、滴定酸度等为指标,研究菊花水煎液对长双歧杆菌生长的影响,并用K—B琼脂法初步研究菊花水煎液对几种常见肠道菌群的抑／促菌作用。结果在5％添加菊花水煎液的培养基中长双歧杆菌的菌体浓度（A600值及活菌计数）均明显高于空白对照,菌液pH也均低于空白对照;长双歧杆菌发酵含有菊花水煎液的牛奶后,凝乳时间缩短,乳液的滴定酸度高于空白对照,pH也相应降低。菊花水煎液对几种常见肠道致病菌有较强的抑菌作用。结论菊花水煎液能促进长双歧杆菌的体外增殖与活性,且具有一定的选择性;菊花有可能成为一种良好的双歧因子或益生元制剂的候选物质。     

淡豆豉对人体肠道六种常住菌的调节作用

目的初探淡豆豉对人体肠道6种常住菌的调节作用。方法利用选择性培养基从人的粪便中分离培养肠杆菌、肠球菌、双歧杆菌、乳杆菌、产气荚膜梭菌和脆弱拟杆菌6种人体肠道常住菌。将高、中和低浓度淡豆豉药液加入选择性培养基中,以不加药液的培养基作为空白对照,根据各菌种培养条件分别于厌氧或好氧条件下培养,然后进行菌落计数。结果与空白对照相比,加药培养基脆弱拟杆菌增长20.00%以上;而其他5种菌的生长均被不同程度的抑制,其中对乳杆菌和肠球菌抑制作用最强,加药培养基上这2种菌均不生长;50.00%浓度药液对产气荚膜梭菌抑制率为32.81%;20.00%浓度药液对肠杆菌完全抑制,但随着药液浓度增大抑制作用减弱,50.00%和70.00%浓度药液对肠杆菌的抑制率分别为53.83%和22.11%;低、中和高浓度药液对双歧杆菌的抑制率分别为17.07%、17.83%和17.84%,差异无统计学意义。结论淡豆豉对人体6种肠道常住菌具有不同程度的调节作用。     

Phytase activity as a novel metabolic feature in Bifidobacterium

Phytase activity has been detected for the first time in Bifidobacterium spp. These bacteria were able to dephosphorylate phytic acid (myo-inositol hexaphosphate, IP(6)) and generate several myo-inositol phosphate intermediates (IP(3)-IP(5)). B. globosum and B. pseudocatenulatum were optimally active at neutral-alkaline pH and B. adolescentis, B. angulatum and B. longum at acid pH. B. pseudocatenulatum showed the highest levels of phytase activity. This species produced maximum activity in the exponential phase of growth and when fructo-oligosaccharides were used as carbon source in the culture medium. The potential role of phytase activity from Bifidobacterium spp. in the reduction of the antinutritional properties of IP(6) is discussed.     

SIMPLE AND RAPID METHOD FOR SCREENING ANTIMICROBIAL ACTIVITIES OF BIFIDOBACTERIUM SPECIES OF HUMAN ISOLATES

The objective of this research was to develop a rapid procedure for the screening antimicrobial activities of Bifidobacterium species of human isolates. A bifidobacteria selective medium BIM-25 agar was modified by adding of 0.5 g/L cysteine-hydrochloride, 1.5 g/L lithium chloride, 1.0 g/L beef extract, and 5 mL/L Tween 20. This medium was inoculated (45C) with diluted fecal material from 5 subjects and overlaid into 0.1% Tween 20 BHI agar plates. Plates were incubated in anaerobic chamber at 37C for 48 h. Plates were then inverted to allow the two layers of agar to fall into the petri lid. BHI soft agar (0.45%) containing Micrococcus luteus (as indicator) was overlaid onto the other layers in the petri dish. Plates were incubated at 37C overnight and zone of growth inhibition was observed. This method is simple and rapid whereas the original method for screening of antimicrobial activities of bifidobacteria is a more time consuming and cumbersome procedure.     

双歧因子对双歧杆菌增殖的影响

应用含有不同浓度的双歧因子(微维乐)的培养基对双歧杆菌进行了定性、定量观察。其结果表明,微维乐对双歧杆菌有明显的促进作用;在双歧杆菌制剂的生产过程中,添加一定量的微维乐,不仅可以提高双歧杆菌收率,而且还可以缩短菌种发酵时间。最佳添加量为1.5%,最佳发酵时间为1.5 h。     

Reduction of vitamin K concentration by salivary Bifidobacterium strains and their possible nutritional competition with Porphyromonas gingivalis

AIMS: To assess the possibility that bifidobacteria compete with Porphyromonas gingivalis for their mutual growth factor vitamin K. This study also examined whether salivary Bifidobacterium species decrease vitamin K concentration in the growth medium. METHODS AND RESULTS: Sixty-five strains of Bifidobacterium were obtained from 20 of 24 periodontally healthy subjects. Bifidobacterium dentium was most frequently detected in the saliva of subjects, followed by Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium urinalis. The growth of most Bifidobacterium isolates, except that of B. urinalis, was stimulated by vitamin K. Moreover, the isolates were capable of decreasing vitamin K after incubation, which suggests that bifidobacteria compete with P. gingivalis for vitamin K. In a co-culture, a representative strain -B. adolescentis S2-1 - inhibited the growth of P. gingivalis if it was inoculated in the medium before P. gingivalis. CONCLUSIONS: B. adolescentis S2-1 decreased vitamin K concentration and inhibited the growth of P. gingivalis by possibly competing for the growth factor. SIGNIFICANCE AND IMPACT OF THE STUDY: Salivary bifidobacteria may possess the potential to suppress the growth of P. gingivalis by reducing the growth factor(s) in the environment.     

The evaluation of a mupirocin-based selective medium for the enumeration of bifidobacteria from probiotic animal feed

In this study, MRS medium supplemented with cysteine hydrochloride and mupirocin, termed Bifidobacterium selective medium (BSM) was found to be elective for bifidobacteria but inhibitory to a wide range of non-bifidobacteria strains commonly included in probiotic animal feed. Bacilli, lactobacilli, lactococci and streptococci failed to form colonies on BSM and enterococci, pediococci and propionibacteria formed colonies <0.5 mm in diameter. Bifidobacteria formed colonies >1 mm in size and could be readily distinguished. The addition of nystatin to BSM further inhibited Saccharomyces cerevisiae. BSM was successfully used to enumerate the bifidobacteria components, confirmed through fructose-6-phophate-phosphoketolase detection, present in two commercial probiotic feeds. The medium is recommended for the enumeration of bifidobacteria from animal feeds especially when not a numerically dominant component.     

A New Plating Medium for the Isolation of Enteric Pathogens: I. Hektoen Enteric Agar

A new agar plating medium for the isolation of enteric pathogens is described. This medium contains greater quantities of peptone to offset the inhibitory effects of bile salts. Additional carbohydrates and larger quantities of those previously used have also been incorporated into the medium to differentiate pathogens from some of the slow lactose fermenters. The use of an indicator system never used before in a differential plating medium opens new possibilities for improvement over traditional and newer selective media. Known strains from the various genera of Enterobacteriaceae have been tested for colonial differentiation as well as for possible inhibitory effects. The results show the medium to be highly selective for the shigellae as well as for other enteric pathogens.     

长双歧杆菌发酵培养基的优化

目的对长双歧杆菌液态发酵培养基进行优化。方法以长双歧杆菌（Bifidobacteriumlongum）为发酵菌株,以MRS培养基为基础培养基,以发酵液活菌数为指标,通过单因素添加实验考察发酵培养基的碳源和氮源的种类,并验证优化后培养基的效果。结果优化后培养基的最适碳源为葡萄糖,最适氮源为酪蛋白胨、牛肉蛋白胨、水解乳蛋白,发酵液活菌数达到2．09×10^9CFU／mL,比原MRS培养基（1．22×10^9CFU／mL）提高了71．30％。结论优化后培养基优于原MRS基础培养基,可应用于长双歧杆菌的液态发酵。     

Identification of plant-associated enterococci

AIMS: To employ an in vitro screening regime to select a probiotic Bifidobacterium strain to complement resistant starch (Hi-maizetrade mark) in a synbiotic yoghurt. METHODS AND RESULTS: Of 40 Bifidobacterium isolates examined, only B. lactis Laftitrade mark B94 possessed all of the required characteristics. This isolate hydrolysed Hi-maizetrade mark, survived well in conditions simulating passage through the gastrointestinal tract and possessed technological properties suitable for yoghurt manufacture. It grew well at temperatures up to 45 degrees C, and grew to a high cell yield in an industrial growth medium. In addition to resistant starch, the organism was able to utilize a range of prebiotics including inulin, and fructo-, galacto-, soybean- and xylo-oligosaccharides. Pulse field gel electrophoresis of restriction enzyme cut chromosomal DNA revealed that B. lactis Laftitrade mark B94 was very closely related to the B. lactis Type Strain (DSM 10140), and to the commercial strains B. lactis Bb-12 and B. lactis DS 920. However, B. lactis Laftitrade mark B94 was the only one of these isolates that could hydrolyse Hi-maizetrade mark. This phenotypic difference did not appear to be due to the presence of plasmid encoded amylase. Bifidobacterium lactis Laftitrade mark B94 survived without substantial loss of viability in synbiotic yoghurt containing Hi-maizetrade mark during storage at 4 degrees C for six weeks. CONCLUSION: Bifidobacterium lactis Laftitrade mark B94 is a promising new yoghurt culture that warrants further investigation to assess its probiotic potential. SIGNIFICANCE AND IMPACT OF THE STUDY: In vitro screening procedures can be used to integrate complementary probiotic and prebiotic ingredients for new synbiotic functional food products.     

Report of the "Bioterrorism Workshop." Duke University Thomas Center on April 2-4, 2002, organized by US Army Research Office

Bifidobacterium spp. have been proposed as a microbial indicator of faecal pollution in water and as possible probiotic in fermented dairy products. These anaerobic bacteria can be subject to metabolic stress in environmental samples because of the presence of oxygen or its derivatives. In this case, their recovery is compromised, especially in selective media. Three reducing agents were tested to improve the recovery of oxygen-stressed bifidobacteria: L-cysteine, sodium pyruvate and sodium thioglycolate. These agents were evaluated individually at various concentrations, and in combination by measuring recovery on rich and selective media. The addition of several mixtures of reducing agents to the water samples and pre-incubation for 4 h at 37 degrees C increased the recovery of Bifidobacterium spp. on culture media.