Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium species

Fecal microbiota of 31 breast-fed, 26 mix-fed, and 11 bottle-fed infants were analyzed by using terminal restriction fragment length polymorphism (T-RFLP), and culture method. We first determined the total and cultivated bacterial counts in infant fecal microbiota. Only approximately 30% of bacteria present in fecal microbiota were cultivable while the remainder was yet-to-be cultured bacteria. Sixty-eight fecal samples were divided into two clusters (I and II) by T-RFLP analysis, and then subdivided into five subclusters (Ia, Ib, IIa, IIb and IIc). There was no clear relationship between clusters and feeding method. A proportion of bifidobacteria was detected in the fecal material by PCR method using species-specific primers. The predominant Bifidobacterium spp. was Bifidobacterium longum longum type (43 samples (63.2%)), followed by B. longum infantis type (23 samples (33.8%)) and B. breve (16 samples (23.5%)). The distribution of Bifidobacterium spp. was similar in the three feeding groups. In contrast, the high incidence of B. breve in cluster I, especially subcluster Ia and B. longum longum type in cluster II, especially subcluster IIa and IIc were characterized by T-RFLP method. Our results showed that the colonization of Bifidobacterium spp. in infant feces correlated with the T-RFLP clusters.     

Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR

To determine the influence of either exclusive breast-feeding or formula feeding on both composition and quantity of the gut microbiota in infants, we have developed real-time, quantitative PCR assays for the detection of Bifidobacterium spp. and Clostridium difficile. Furthermore, we have monitored the prevalence and counts of Escherichia coli by applying a previously described real-time PCR assay. We found all 100 infants tested to be colonized by Bifidobacterium spp. The bifidobacterial counts were comparable between the 50 breast-fed and 50 formula-fed infants with median values of 10.56 log10 and 10.24 log10 CFU g(-1) wet weight faeces, respectively. C. difficile was detected in 14% of the breast-fed and 30% of the formula-fed infants. In addition, the C. difficile counts were significantly lower in breast-fed infants than in the formula-fed group (median values of 3.28 log10 and 7.43 log10 CFU g(-1), respectively; p=0.03). The prevalence of E. coli in the breast-fed and formula-fed group was 80% and 94%, respectively. Also, the E. coli counts in colonized infants was significantly lower in the breast-fed infants than in the formula-fed group (median values of 9.11 log10 and 9.57 log10 CFU g(-1), respectively; p=0.004). We conclude that the prevalence and counts of C. difficile as well as E. coli are significantly lower in the gut microbiota of breast-fed infants than in that of formula-fed infants, whereas the prevalence and counts of Bifidobacterium spp. is similar among both groups.     

Growth of various intestinal bacteria on alternansucrase-derived oligosaccharides

AIMS: To determine whether alternansucrase (ASR)-derived oligosaccharides can support the in vitro growth of various intestinal bacteria. METHODS AND RESULTS: Growth was assessed from each culture after incubation in a medium containing ASR-derived oligosaccharide as sole carbohydrate source. Most of the Bifidobacterium spp. tested showed growth on all five of the oligosaccharides tested while the Lactobacillus spp., Bacteroides thetaiotaomicron, coliforms and pathogenic bacteria displayed no or little growth. CONCLUSIONS: The ASR-derived oligosaccharides were selectively utilized by many of the Bifidobacterium spp. tested but did not support significant growth of the Lactobacillus spp., Bact. thetaiotaomicron, coliforms and pathogenic bacteria tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Alternansucrase-derived oligosaccharides are a potential source of new prebiotics.     

Analysis of the large bowel microbiota of colitic mice using PCR/DGGE

AIM: To test combined polymerase chain reaction amplification of 16S rRNA gene sequences and denaturing gradient gel electrophoresis (PCR/DGGE) as an analytical method to investigate the composition of the large bowel microbiota of mice during the development of colitis. METHODS AND RESULTS: The colonic microbiota of formerly germfree interleukin 10 (IL-10)-deficient mice that had been exposed to the faecal microbiota of specific pathogen-free animals was screened using PCR/DGGE. The composition of the large bowel microbiota of IL-10-deficient mice changed as colitis progressed. DNA fragments originating from four bacterial populations ('Bacteroides sp.', Bifidobacterium animalis, Clostridium cocleatum, enterococci) were more apparent in PCR/DGGE profiles of colitic mice relative to non-colitic animals, whereas two populations were less apparent (Eubacterium ventriosum, Acidophilus group lactobacilli). Specific DNA:RNA dot blot analysis showed that bifidobacterial ribosomal RNA (rRNA) abundance increased as colitis developed. CONCLUSIONS: PCR/DGGE was shown to be an effective method to demonstrate changes in the composition of the large bowel microbiota of mice in relation to progression of inflammatory disease. The intensity of staining of DNA fragments in DGGE profiles reflected increased abundance of bifidobacterial rRNA in the microbiota of colitic animals. As bifidobacterial fragments in PCR/DGGE profiles generated from microbiota DNA showed increased intensity of fragment staining, an increase in bifidobacterial numbers in colitic mice was indicated. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR/DGGE analysis demonstrated an altered composition of the large bowel microbiota of colitic mice. This work will allow specific groups of bacteria to be targeted in future research concerning the pathogenesis of colitis.     

Growth of Bifidobacterium longum BB536 in medida (fermented cereal porridge) and their survival during refrigerated storage

AIMS: To develop medida, a Sudanese fermented thin porridge as a probiotic dietary adjunct with high total solids. METHODS AND RESULTS: Fifteen per cent brown rice flour of 2-day-old malted paddy and skim milk were used for formulation. Levels of 2.25, 4.5 and 10% of added skim milk were studied. The initial pH was 6.7 and fermentation was run to a final pH of 4.4 using culture of Bifidobacterium longum BB 536. The highest count of 9.9 +/- 0.07 log CFU ml(-1) was obtained with 10% of added skim milk. The total solids at this level was 21%, 11.1 times more compared with the traditionally prepared medida using un-malted brown rice. The viscosity was low and the flowing characteristic was stable. The final productions of lactic and acetic acids were 56.8 +/- 0.80 and 56.3 +/- 2.00 mumol ml(-1) respectively. The high ratio of acetate to lactate decreased as fermentation continues due to the increase in the rate of lactate production. Under refrigerated storage the count of B. longum BB 536 remained relatively stable during the first week (9.7 +/- 0.10 log CFU ml(-1)) then subsequently decreased by 0.9 log CFU ml(-1) in the following week. CONCLUSIONS: The results of this study demonstrated that fermented medida made from malted brown rice is a suitable food system for the delivery of B. longum BB 536 with a relatively stable shelf life. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first attempt to prepare fermented medida from malted flour with bifidobacteria having the highest total solids while still maintaining the flowing characteristics. Previous studies on medida did not go beyond the use of alpha amylase enzyme and pure lactic acid bacteria isolates from spontaneously fermented dough.     

Production of Propionibacterium shermanii biomass and vitamin B12 on spent media

AIMS: The propionibacteria are commercially important due to their use in the cheese industry, and there is a growing interest for their probiotic effects. Stimulatory effects of lactic acid bacteria (LAB) on propionic acid bacteria have been observed. This study was designed to examine the possibility of using spent media previously used to grow LAB for the production of biomass and metabolites of Propionibacterium freudenreichii subsp. shermanii. METHODS AND RESULTS: Seventeen MRS and vegetable juice media were prefermented by various LAB and evaluated for their ability to subsequently support the growth of Propionibacterium, using automated spectrophotometry (AS). Growth of Propionibacterium in spent media was strongly affected by the LAB strain used to produce the spent medium. The native MRS medium (not prefermented) yielded the highest optical density values followed by prefermented media by Lactobacillus acidophilus, Bifidobacterium longum and Lactococcus lactis. Prefermented cabbage juice enabled good growth of Propionibacterium. For the production of organic acids and vitamin B12, cells of Propionibacterium were concentrated and immobilized in alginate beads in the aim of accelerating the bioconversions. More propionic acid was obtained in spent media than in native MRS. The concentration of vitamin B12 was higher in media fermented with free cells than those with immobilized cultures; with the free cells, its concentration varied from 900 to 1800 ng ml(-1) of media. CONCLUSIONS: It was demonstrated that spent media could be recycled for the production of Propionibacterium and metabolites, depending on the LAB strain that was previously grown. Media remediation is needed to improve the production of vitamin B12, especially with immobilized cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an option for recycling of spent media generated by producers of LAB or producers of fermented vegetables. The propionic fermentation may result in three commercial products: biomass, vitamin B12 or organic acids, which may be used as starters, supplements or food preservatives. It is an attractive process from economical and environmental standpoints.     

口服双歧杆菌,酪酸梭菌活菌制剂药效作用研究

利用氨苄青霉素引起ＳＰＦ－ＢＡＬＢ／Ｃ小鼠肠道菌群失调腹泻作为模型,观察肠康平制调整扬道菌群的药效作用,并与它同类单价制剂不平制剂对该ＳＰＦ－ＢＡＬＢ／Ｃ小鼠肠道菌群失调腹泻具有治疗作用,高剂量组９１０＾６ＣＦＵ／次）的治疗效果优于低剂量组（１０＾４ＣＦＵ／次）,肠康平制剂缓解ＳＰＦ－ＢＡＬＢ／Ｃ小鼠筵菌群失调后腹泻症状的时间较单价制剂略快。     

Gas chromatography-mass spectrometry (GC-MS) techniques for metabolic flux analysis of the Bifido shunt pathway

The presence of the Bifido shunt in Bifidobacterium is predicted to lead to the uptake and metabolic conversion of fructose to acetate and lactate. We propose an approach to quantifying the carbon flux through the Bifido shunt by measuring specific ¹³C-labeled carbohydrate-derived isotopomers by gas chromatography-mass spectrometry (GC-MS). The techniques described may provide an alternative approach for determining the in vitro prebiotic potential of dietary oligosaccharides.     

双歧醋对常见病原菌的生物拮抗作用研究

目的研究双歧醋对常见病原菌的生物拮抗作用及影响因素。方法采用纸片扩散法(K-B法)、琼脂打孔法和试管稀释法(M IC法)测定双歧醋及其处理物(加热、不同pH)对常见病原菌的拮抗作用,并讨论pH、加热对拮抗作用的影响,同时用某市售醋作为对照。结果双歧醋及其处理物均具有一定的抑菌作用。结论双歧醋对常见病原菌有一定的拮抗作用,抑菌作用强于市售醋。     

双歧杆菌活菌胶囊预防CPT-11引起的化疗相关性腹泻的疗效观察

目的观察双歧杆菌活菌胶囊预防伊立替康(CPT-11)引起的化疗相关性腹泻的疗效。方法 36例患者随机分成2组,观察组:化疗+双歧杆菌活菌胶囊,对照组:单纯化疗。观察患者治疗前后的KPS评分和腹泻的发生率及分级。结果观察组化疗前后KPS评分无明显变化,对照组化疗后KPS评分明显下降,差异有统计学意义,观察组Ⅰ～Ⅱ级腹泻发生率为33.3%,对照组为50%,2组差异有统计学意义(P<0.01)。观察组Ⅲ～Ⅳ级腹泻发生率为5.5%,对照组为27.8%,2组差异有统计学意义(P<0.01)。结论应用双歧杆菌活菌胶囊预防化疗相关性腹泻有良好的效果。