Diversity of the gut microbiota and eczema in early life

<p>Abstract</p> <p>Background</p> <p>A modest number of prospective studies of the composition of the intestinal microbiota and eczema in early life have yielded conflicting results.</p> <p>Objective</p> <p>To examine the relationship between the bacterial diversity of the gut and the development of eczema in early life by methods other than stool culture.</p> <p>Methods</p> <p>Fecal samples were collected from 21 infants at 1 and 4 months of life. Nine infants were diagnosed with eczema by the age of 6 months (cases) and 12 infants were not (controls). After conducting denaturating gradient gel electrophoresis (DGGE) of stool samples, we compared the microbial diversity of cases and controls using the number of electrophoretic bands and the Shannon index of diversity (<it>H'</it>) as indicators.</p> <p>Results</p> <p>Control subjects had significantly greater fecal microbial diversity than children with eczema at ages 1 (mean <it>H' </it>for controls = 0.75 vs. 0.53 for cases, P = 0.01) and 4 months (mean <it>H' </it>for controls = 0.92 vs. 0.59 for cases, P = 0.02). The increase in diversity from 1 to 4 months of age was significant in controls (P = 0.04) but not in children who developed eczema by 6 months of age (P = 0.32).</p> <p>Conclusion</p> <p>Our findings suggest that reduced microbial diversity is associated with the development of eczema in early life.</p

Site and Strain-Specific Variation in Gut Microbiota Profiles and Metabolism in Experimental Mice

The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables.The GTM composition of six highly inbred, genetically distinct strains of mouse (C3H, C57, GFEC, CD1, CBA nu/nu and SCID) was profiled using eubacterial -specific PCR-DGGE and quantitative PCR of feces. Animals exhibited strain-specific fecal eubacterial profiles that were highly stable (c. >95% concordance over 26 months for C57). Analyses of mice that had been relocated before and after maturity indicated marked, reproducible changes in fecal consortia and that occurred only in young animals. Implantation of a female BDF1 mouse with genetically distinct (C57 and Agoutie) embryos produced highly similar GTM profiles (c. 95% concordance) between mother and offspring, regardless of offspring strain, which was also reflected in urinary metabolite profiles. Marked institution-specific GTM profiles were apparent in C3H mice raised in two different research institutions.Strain-specific data were suggestive of genetic determination of the composition and activities of intestinal symbiotic consortia. However, relocation studies and uterine implantation demonstrated the dominance of environmental influences on the GTM. This was manifested in large variations between isogenic adult mice reared in different research institutions

Development of new screening techniques to assess bioavailability of soy isoflavones: use of a hamster model and genomic techniques to identify human fecal microorganisms associated with isoflavone disappearance

Isoflavones are found almost exclusively in soybeans and soy products. They have been extensively studied for improving health. Better understanding isoflavones bioavailability is also essential compared to their metabolism. Two aspects of isoflavone bioavailability were investigated in this project;Golden Syrian hamsters were investigated to attempt validating this model as relevant to humans for isoflavones bioavailability and their cholesterol lowering effect. Differences between sex (females\u3emales) and isoflavone (genistein\u3edaidzein), similar patterns of urinary excretion between females and women and clustering of animals as high excreters/low degraders and low excreters/high degraders for daidzein and genistein were features shared with humans that may validate hamsters as a model to study isoflavones bioavailability. Cecal but not fecal in vitro daidzein and genistein degradation rates were correlated with urinary excretion levels. Finally, high daidzein, genistein and total isoflavone urinary excretion was associated with lower levels of total cholesterol compared to low excretion;To identify human fecal bacterial species influencing isoflavone disappearance, polymerase chain reaction and denaturing gradient gel electrophoresis allowed qualitative and significantly quantitative profiling of fecal microbial profiles based on each strain\u27s 16S DNA. When comparing gut microbial profiles to in vitro fecal isoflavones disappearance, 7 bands of interest were identified when focusing on intra-individual variability of fecal glycitein degradation over a one month interval. Screening of 33 subjects gave 5 bands of greater intensity in the 4 highest compared to the 4 lowest glycitein degraders, 5 bands for genistein and no bands for daidzein. Sequencing and identification of microorganisms using Basic Local Alignment Search Tool gave matches to Bacteroides, Prevotella and Clostridium species. These strains were investigated using a fecal suspension matrix where nutrient availability was rich (attempting to mimic rapid gut transit time and low degradation rate) or poor (long gut transit time; high degradation rate). Species increasing significantly isoflavones disappearance in both media were B. acidifaciens, T. forsythensis, P. pallens, B. uniformis, E. ramulus and C. orbiscindens. These species were hypothesized to be present all individuals with greater proportions of these strains in high versus low degraders, as reflected by DGGE analysis

Changes in Human Fecal Microbiota Due to Chemotherapy Analyzed by TaqMan-PCR, 454 Sequencing and PCR-DGGE Fingerprinting

BACKGROUND: We investigated whether chemotherapy with the presence or absence of antibiotics against different kinds of cancer changed the gastrointestinal microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Feces of 17 ambulant patients receiving chemotherapy with or without concomitant antibiotics were analyzed before and after the chemotherapy cycle at four time points in comparison to 17 gender-, age- and lifestyle-matched healthy controls. We targeted 16S rRNA genes of all bacteria, Bacteroides, bifidobacteria, Clostridium cluster IV and XIVa as well as C. difficile with TaqMan qPCR, denaturing gradient gel electrophoresis (DGGE) fingerprinting and high-throughput sequencing. After a significant drop in the abundance of microbiota (p = 0.037) following a single treatment the microbiota recovered within a few days. The chemotherapeutical treatment marginally affected the Bacteroides while the Clostridium cluster IV and XIVa were significantly more sensitive to chemotherapy and antibiotic treatment. DGGE fingerprinting showed decreased diversity of Clostridium cluster IV and XIVa in response to chemotherapy with cluster IV diversity being particularly affected by antibiotics. The occurrence of C. difficile in three out of seventeen subjects was accompanied by a decrease in the genera Bifidobacterium, Lactobacillus, Veillonella and Faecalibacterium prausnitzii. Enterococcus faecium increased following chemotherapy. CONCLUSIONS/SIGNIFICANCE: Despite high individual variations, these results suggest that the observed changes in the human gut microbiota may favor colonization with C. difficile and Enterococcus faecium. Perturbed microbiota may be a target for specific mitigation with safe pre- and probiotics

New Insights into Human Gut Microbiota Development in Early Infancy : Influence of Diet, Environment and Mother’s Microbiota

The human gut is home to an enormous number of microbes that play an important role in host physiology, metabolism, nutrition and immune function. Deviations in initial human gut microbiota development have been associated with a number of immune-mediated diseases including inflammatory bowel diseases, atopy and allergies, as well as obesity, which are reaching epidemic proportions in the westernized world. However, gut microbiota dysbiosis has also been linked to health problems common in the developing world including malnutrition. The aim of this thesis was to unravel the impact of specific factors, including environment, geographical location, diet and mother’s microbiota, on the gut microbiota development in early infancy. Analysis of gut microbiota composition of six-month-old infants from Malawi and Finland, representing developing and westernized countries, revealed that the Malawians had a distinct Lactobacillus microbiota that was richer and more diverse compared to their Finnish counterparts. Interestingly, we detected L. rhamnosus GG, a widely used probiotic in Finland, in a third of the Finnish infants even though the infants or their mothers did not receive any probiotic products. For the detection, we used a L. rhamnosus GG strain-specific detection system, whose specificity was confirmed in this thesis. In addition, we noted that lipid-based nutrient supplements do not have an impact on gut microbiota development in Malawian infants. These dietary supplements have been used in several clinical trials to promote growth of children from developing countries, but their impact on the gut microbiota has been previously unknown. In addition to environment and diet, a number of host factors may have an impact on the gut microbiota development. Here, we assessed how mother’s microbiota influences the gut microbiota development in early infancy by comparing samples of maternal feces, placenta, amniotic fluid, colostrum, meconium and infant feces from mother-infant pairs. Our results suggest that the microbial colonization may begin already during fetal life. After birth, colostrum seems to influence the gut microbiota development of the neonate by serving as a source of microbes and microbiota modulating factors, such as human milk oligosaccharides. We also noted that human milk oligosaccharides and microbiota of the colostrum are interrelated. Taken together, this thesis provides further knowledge on how different factors influence gut microbiota development in early life. Modification of gut microbiota composition during its development may be a viable strategy to prevent or treat inflammatory non-communicable diseases in the future.Ihmisen suolistossa asuu valtava määrä mikrobeja, jotka vaikuttavat merkittävästi ihmisen fysiologiaan, metaboliaan, ravitsemukseen ja immunijärjestelmän toimintaan. Suoliston mikrobiston kehityksen häiriöiden on todettu olevan yhteydessä lukuisiin immuunivälitteisiin sairauksiin, kuten tulehduksellisiin suolistosairauksiin, atopiaan, allergioihin ja liikalihavuuteen. Nämä sairaudet ovat yleistyneet etenkin länsimaisissa hyvinvointivaltioissa viime vuosikymmeninä. Lisäksi viime aikoina on havaittu, että suoliston mikrobiston häiriöt ovat yhteydessä kehittyvissä maissa yleisiin sairauksiin kuten aliravitsemukseen. Tämän väitöskirjatyön tavoitteena oli tutkia eri tekijöiden kuten ympäristön, maantieteellisen sijainnin, ruokavalion ja äidin mikrobiston vaikutusta suoliston mikrobiston kehitykseen varhaislapsuudessa. Malawilaisten ja suomalaisten kuuden kuukauden ikäisten lasten suolistomikrobiston analyysi osoitti, että malawilaisilla oli rikkaampi ja monipuolisempi Lactobacillus –mikrobisto, jonka koostumus oli selkeästi erilainen kuin suomalaisilla. Tutkimuksessa havaittiin yllättäen, että kolmasosalla suomalaisista lapsista oli L. rhamnosus GG probioottia suolistossa, vaikka lapset tai äidit eivät olleet nauttineet probioottituotteita. Havainto tehtiin käyttäen L. rhamnosus GG laji-spesifistä PCR-pohjaista menetelmää, jonka tarkkuus arvioitiin ja todettiin hyväksi tässä väitöskirjatyössä. Lisäksi osoitettiin, että rasvapohjaiset ravintolisät eivät vaikuta malawilaisten lasten suolistomikrobiston kehitykseen. Rasvapohjaisilla ravintolisillä on pyritty ehkäisemään lasten aliravitsemusta kehitysmaissa, mutta niiden vaikutusta suoliston mikrobistoon ei ole aikaisemmin tunnettu. Ympäristön ja ruokavalion lisäksi monet isännästä riippuvat tekijät voivat vaikuttaa suoliston mikrobiston kehitykseen. Tässä työssä selvitettiin äidin mikrobisto vaikutusta lapsen mikrobiston kehitykseen varhaislapsuudessa vertailemalla mikrobistonäytteitä äidin ulosteesta, istukasta, lapsivedestä, äidinmaidosta, mekoniumista, sekä lapsen ulosteesta. Tulokset osoittivat, suoliston kolonisaatio alkaa mahdollisesti jo ennen syntymää kohdussa. Tämän jälkeen lapsen mikrobiston kehitykseen vaikuttavat rintamaidon sisältämät mikrobit ja mikrobistoa muokkaavat tekijät, kuten äidinmaidon oligosakkaridit. Työssä havaittiin myös, että äidinmaidon oligosakkaridit ja mikrobisto ovat yhteyksissä toisiinsa. Tämä väitöskirjatyö tuotti uutta tietoa siitä kuinka eri tekijät vaikuttavat suolistomikrobiston kehitykseen. Suoliston mikrobiston muokkaaminen kehityksen eri vaiheissa voi olla yksi toimiva vaihtoehto tulehduksellisten ei-tarttuvien sairauksien ehkäisemiseen ja parantamiseen tulevaisuudessa.Siirretty Doriast

Gut Microbiota Dysbiosis Associated With Altered Production of Short Chain Fatty Acids in Children With Neurodevelopmental Disorders

While gut microbiota dysbiosis has been linked with autism, its role in the etiology of other neurodevelopmental disorders (NDD) is largely underexplored. To our knowledge this is the first study to evaluate gut microbiota diversity and composition in 36 children from the Republic of Serbia diagnosed with NDD and 28 healthy children. The results revealed an increased incidence of potentially harmful bacteria, closely related to Clostridium species, in the NDD patient group compared to the Control group: Desulfotomaculum guttoideum (P lt 0.01), Intestinibacter bartlettii (P lt 0.05), and Romboutsia ilealis (P lt 0.001). On the other hand, significantly lower diversity of common commensal bacteria in the NDD group of patients was noticed. Enterococcus faecalis (P lt 0.05), Enterococcus gallinarum (P lt 0.01), Streptococcus pasteurianus (P lt 0.05), Lactobacillus rhamnosus (P lt 0.01) and Bifidobacteria sp. were detected in lower numbers of patients or were even absent in some NDD patients. In addition, butyrate-producing bacteria Faecalibacterium prausnitzii (P lt 0.01), Butyricicoccus pullicaecorum (P lt 0.05), and Eubacterium rectale (P = 0.07) were less frequent in the NDD patient group. In line with that, the levels of fecal short chain fatty acids (SCFAs) were determined. Although significant differences in SCFA levels were not detected between NDD patients and the Control group, a positive correlation was noted between number of rDNA amplicons obtained with universal primers and level of propionic acid, as well as a trend for levels of total SCFAs and butyric acid in the Control group. This correlation is lost in the NDD patient group, indicating that NDD patients' microbiota differs from the microbiota of healthy children in the presence or number of strong SCFA-producing bacteria. According to a range-weighted richness index it was observed that microbial diversity was significantly lower in the NDD patient group. Our study reveals that the intestinal microbiota from NDD patients differs from the microbiota of healthy children. It is hypothesized that early life microbiome might have an impact on GI disturbances and accompanied behavioral problems frequently observed in patients with a broad spectrum of NDD

Assessing prebiotic effects of resistant starch on modulating gut microbiota with an in vivo animal model and an in vitro semi-continuous fermentation model

Resistant starch (RS) is the starch that escapes human digestion system and will be delivered to the lower gut for fermentation by gut microbiota. Five types of resistant starch have been described, including 1) physically amylase-inaccessible type 1 RS, 2) native high amylose type 2 RS, 3) retrograded type 3 RS, 4) chemically modified type 4 RS, and 5) fatty acid complexed type 5 RS. RS has been shown to lower postprandial glycemic index (including plasma glucose and insulin levels), indicating effects of RS in reducing risk of type II diabetes and possible obesity as well, In addition, RS fermentation in the lower gut has been shown to maintain colon health and prevent colon carcinogenesis, mainly through production of SCFA. Physiological significances of SCFA, especially butyrate, include providing an energy source for colonocytes, maintaining the colonic epithelia cell layer, stimulating basal crypt cell proliferation, and inducing apoptosis of hyper-proliferative cancer cells. Furthermore, RS has also been considered as a prebiotic to modulate gut microbiota favoring RS degradation and SCFA production. The long-term objective of our study is to understand the underlying mechanisms of RS in improving human health from two correlated aspects, modulating gut microbiota and producing SCFA. We hypothesized that when using an in vivo animal model and an in vitro fermentation model, resistant starch consumption would shift gut microbiota pattern towards favoring RS fermentation, especially SCFA production, In respect to the physiological significance of resistant starch implicated in improving colon health, we hypothesized that HA7 (high amylose starch VII, type 2 RS), and HA7-SA (palmitic acid complexed HA7, type 5 RS) would modulate colonic microbiota into different patterns. The bacterial pattern shifts can explain the differential efficacy of two RS suppressing colon carcinogenesis. Previous studies showed that pre-neoplasm, aberrant crypt foci (ACF) induced by carcinogen Azoxymethane (AOM) were only decreased by 8-wk feeding of HA7 (21 y 9) in male Fisher 344 rats, but not by HA7-SA (38 y 18), when compared to highly digested control starch (48 y 14). We used PCR-DGGE (Denaturing Gradient Gel Electrophoresis) to analyze colonic microbiota with bacteria universal 16S rRNA gene primers. Our results showed two RSs induced significant shifts of colonic microbiota compared to control digestible starch-fed rats. Moreover, differential bacterial patterns were observed between two RSs such as the specific enrichment of putative Bifidobacterium pseudolongum by HA7-SA, but not by HA7. More importantly, a significant correlation was observed between gut microbiota patterns and ACF numbers developed in AOM treated animals. Further analysis of starch fermentation-associated bacteria groups showed similar shifts of Clostridium cluster IV by two RSs with increased putative Ruminococcus bromii. By contrast, the bacterial pattern of Clostridium cluster XIVa showed correlation with ACF number. Specific enrichment of putative Ruminococcus obeum R. sp. SR1/5 was observed in HA7-fed animals, whereas specific decrease of Bacteroides sp. ASF519 or Parabacteroides goldsteinii (T) by HA7-SA was observed in the Bacteroides fragilis group. Our results suggested that gut microbiota patterns modulated by RS were related to differential efficacy of HA7 and HA7-SA in decreasing colonic carcinogenesis. We established an in vitro semi-continuous anaerobic incubation model to compare fermentability, i.e. SCFA production, of four high amylose starches: HAV, HAVI, HAVII and GEMS-067 from four maize lines with different genetic background and amylose contents (55%, 65%, 70% and 70% respectively). This was done to evaluate prebiotic effects of resistant starch on modulating gut microbiota hypothetically towards favoring RS fermentation. The digested starch residues (SR), obtained from in vitro digestion with the AOAC 991.43 method, were incubated with fecal microbiota to simulate human digestion of cooked starchy food. In our study a total of 32 individuals were recruited: 17 lean, 9 overweight and 6 obese individuals. The study was conducted in two phases 5 months apart. Each phase consisted of a 3-wk incubation period with a frequency of changing BHI (Brain Heart Infusion) medium and SR substrates every 3.5 d. We observed significantly decreased pH, increased gas production, increased butyrate and total SCFA concentration in incubations with the four SR compared to blank BHI medium control starting at wk 1. There was no difference between SR. Molar proportions of butyrate was increased by SR with decreased acetate proportion, both of which achieved stability starting at wk 2. Additionally, propionate concentration was only increased by SRV at the end of the 3-wk incubation compared to BHI medium, but not by other SR. Large inter-individual variation was observed in the proportional increase of butyrate by SR compared to blank BHI medium control. No significant difference was found between lean and overweight individuals from fermentation indicators measured. We concluded that a stable long-term semi-continuous in vitro fermentation model was established to simulate carbohydrate fermentation in human lower gut. We also showed significant increase of butyrate production by RS fermentation with human fecal microbiota. The modulation of microbiota by amylase digestion residues of HAV, VI and VII in our semi-continuous fermentation model was further analyzed with PCR-DGGE by examining total bacterial pattern and that of RS fermentation associated bacteria groups, including Clostridium cluster IV, XIVa and Bacteroides fragilis group. We hypothesized that bacterial patterns obtained after 3 wk fermentation will be selected by three SR incubation and lean/overweight microbiota as well. Our results showed that total bacterial patterns were shifted by three SR incubation with fecal microbiota from 30 donors at the end of 3-wk fermentation compared to control BHI medium. Moreover, bacterial pattern in SRV fermentation samples differed from that of SRVI and SRVII, which shared a certain degree of similarity. However, bacterial pattern of Clostridium cluster IV and XIVa in SRVI fermentation samples differed from SRV and SRVII, which shared similar patterns, whereas no shifts were observed by any SR in Bacteroides fragilis group bacterial pattern compared to blank BHI medium control. Most important, we observed the putative Ruminococcus bromii was specifically selected during SR incubation by microbiota from lean individuals, but not by microbiota from overweight and obese individuals. We concluded that our in vitro semi-continuous fermentation model can be used to assess prebiotic effects of RS by simulating long-term RS consumption in human. We also showed that Ruminococcus bromii, belonging to Clostridium cluster IV, was selectively enriched by SR and microbiota of lean individuals. In summary, our studies with both in vivo animal model and in vitro fermentation model supported previous recognition of resistant starch acting as a prebiotic to modulate gut microbiota, especially on Clostridium clusters IV and XIVa. Ruminococcus bromii was specifically induced in rat model fed RS as well as in vitro fermentation model using lean microbiota. Moreover, different RS may have different fermentation outcomes. Our findings provided solid evidence to answer the fundamental question of how RS exerted effects on shifting bacterial pattern. In addition, we also showed that the physiological significance of RS might be affected by physical-chemical properties of starch and pre-existing microbiota as well

Dietary effects on human fecal microbiota

The establishment of microbial populations in the gastrointestinal (GI)-tract is a complex process, involving microbial and host interactions eventually resulting in a dense and stable population. Recently, the identification of microbial species from fecal samples has become more accurate with the use of 16S RNA gene-based methods. However, although these molecular-based detection methods have apparent benefits over culture-based techniques, they involve potential pitfalls that should be taken into consideration when studying the fecal microbiota, such as the storage conditions and deoxyribonucleic acid (DNA)-extraction. Therefore, the effects of different storage conditions and DNA-extraction protocols on fecal samples were evaluated in this study. Whereas the DNA-extraction protocol did not affect the numbers of Bac-teroides spp., the abundance of this group showed a significant decrease after one week s storage at -20°C. Furthermore, the numbers of predominant bacteria, Eubacterium rectale group, Clostridium leptum group, bifidobacteria and Atopobium group, were significantly higher in samples stored at -70°C after mechanical DNA-extraction than after enzymatic DNA-extraction as detected with real-time PCR (qPCR). These results indicate that rigorous mechanical lysis leads to the detection of higher bacterial numbers from human fecal samples than enzymatic DNA-extraction. Therefore, the use of different DNA-extraction protocols may partly ex-plain contradictory results reported in previous studies. The composition of the human intestinal microbiota is influenced by host-specific factors such as age, genetics and physical and chemical conditions encountered in the GI-tract. On the other hand, it is modulated by environmental factors with impact on the host during the lifespan, such as diet. The impact of diet on the gut microbiota has usually been assessed by subjecting people to the same controlled diet, and thereafter following the shifts in the microbiota. In the present study, the habitual dietary intake of monozygotic twins was associated with the fecal microbiota com-position, which was analysed using qPCR and Denaturing Gradient Gel Electrophoresis (DGGE). The effect of diet on the numbers of the bacteria was described using a hierarchical linear mixed model that included the twin individuals, stratified by body mass index, and their families as random effects. The abundance and diversity of the bacterial groups studied did not differ between normal weight, overweight, and obese individuals with the techniques used. However, intakes of energy, monounsaturated fat, (n-3) polyunsaturated fat, (n-6) polyunsaturated fat and soluble fibre had significant associations with the fecal bacterial numbers. In addition, co-twins with identical energy intakes had more similar numbers and DGGE-profile diversities of Bacteroides spp. than co-twins with different intakes. Moreover, co-twins who ingested the same amounts of saturated fat had very similar DGGE-profiles of Bacteroides spp., whereas co-twins with similar consumption of fibre had very low bifidobacterial DGGE-profile similarity. Thereafter, the impact of the energy intake on the fecal microbiota of a group of 16 obese individuals was assessed during a 12 month intervention, which consisted of a 6 week very low energy diet (VLED) and thereafter a follow-up period of 5, 8 and 12 months. The diet plan was combined with exercise and lifestyle counseling. Fecal samples were analyzed using qPCR, DGGE and fluorescent in situ hybridization. The effect of the energy restricted diet on the fecal bacterial numbers was described using a linear mixed model that accounted for repeated measurements in the same individual. The VLED period affected the major fecal microbial groups; in particular bifidobacteria decreased compared to the baseline numbers. Furthermore, the change in numbers of the fecal bacterial groups studied, followed the dietary intake and not the weight changes during the 12 months. Methanogens were detected in 56% of the participants at every sampling time point, regardless of the change in dietary intake. In addition, weight loss was associated with a decrease in Lactobacillus group bacteria. These findings confirm that the diet and energetic intake play an important role in modulation of the fecal microbiota. Finally, the potential of utilising the information on expression levels of selected stress genes in assessing the quality of probiotic products was evaluated. For this purpose, reverse transcription-qPCR methods were developed to study the expres-sion of clpL1 and clpL2 stress genes in Lactobacillus rhamnosus VTT E-97800 cells after exposure to processing-related stress conditions or to freeze-drying. Heat treatments were performed with L. rhamnosus VTT E-97800 in laboratory scale, whereas acid treatments were performed both in laboratory and fermenter scale. RNA was extracted from fresh cells and freeze-dried powders. clpL1 and clpL2 transcripts were analysed by qPCR using SYBR Green I. clpL1 was induced in L. rhamnosus VTT E-97800 cells exposed to 50°C and to a much lesser extent in cells exposed to 47°C. No induction was observed for clpL2 during either acid or heat treatment in any of the conditions applied. RNA isolation from freeze-dried powders was unsuccessful, although several attempts were made with high quality products. These results suggest that developing quality indicators for probiotic prod-ucts based on differences in the expression of stress genes will be a challenging task, since rather harsh conditions are apparently needed to detect differences in the gene expression. In addition, the unsuccessful RNA isolation from freeze-dried pow-ders hampers the applicability of this technique in the quality control of probiotic products.Perustaminen mikrobipopulaatioidenruoansulatuskanavan (GI)- suolikanavan onmonimutkainen prosessi , johon mikrobi-ja isännän vuorovaikutukset lopulta tuloksenatiivis ja vakaa väestöstä. Äskettäin tunnistaminen mikrobi- lajien ulostenäytteet on tullut tarkempi käytön 16S RNA-geeniä - menetelmiä . Vaikka nämä molekyylipohjaisia ​​analyysimenetelmiä on ilmeistä hyötyä yli kulttuuri - tekniikat , niihin liittyy mahdollisia sudenkuoppia , jotka olisi otettava huomioon , kun tutkitaanulosteen mikrobiston , kutensäilytysolosuhteet ja deoksiribonukleiinihappo ( DNA ) - uutto . Siksivaikutuksia eri säilytysolosuhteet ja DNA - uutto pöytäkirjat ulostenäytteet tutkittiin tässä tutkimuksessa . Ottaa huomioon, että DNA - uutto -protokollaa ei vaikuttanut numerot Bac - teroides spp . ,Runsaasti tämä ryhmä osoittimerkittävää laskua yhden viikon varastoinnin -20 ° C: ssa Lisäksinumerot hallitseva bakteereja , Eubacterium rectale ryhmä , Clostridium leptum ryhmä , bifidobakteerien ja Atopobium ryhmä , olivat huomattavasti korkeammat näytteitä säilytetään -70 ° C mekaanisen DNA - uutto kuin entsymaattisen DNA - uutto havaita reaaliaikainen PCR- ( qPCR ) . Nämä tulokset osoittavat, että tiukka mekaaninen hajoaminen johtaahavaitseminen bakteereihin ihmisen ulostenäytteissä kuin entsymaattinen DNA - uutto . Siksierilaisten DNA - uutto protokollia osittain ex - tavallinen ristiriitaisia ​​tuloksia raportoitu aiemmissa tutkimuksissa . Koostumusihmisen suoliston mikrobisto vaikuttavat Isäntäspesifinen tekijät, kuten ikä, perimä ja fysikaaliset ja kemialliset olosuhteet kohdataanGI - suolikanavan . Toisaalta , se moduloidaan ympäristötekijät , jotka vaikuttavatisäntä elinkaaren aikana , kuten ruokavalio . Ruokavalion vaikutussuoliston mikrobiston on yleensä arvioitu alistamalla ihmisiäsamaan ruokavaliota , ja sen jälkeen kunmuutoksiamikrobiston . Tässä tutkimuksessa ,vakinainen ravinnosta monozygotic kaksoset liittyiulosteen mikrobiston com - asema , joka analysoitiin qPCR ja Denaturing Gradient (DGGE ) . Ruokavalion vaikutustanumerotbakteerien kuvattiinhierarkkinen lineaarinen yhdistettyä mallia , joka sisälsikaksi erillistä yksilöitä , ositettu painoindeksi ja heidän perheidensä satunnaisia ​​vaikutuksia . Runsauteen ja monimuotoisuuteenbakteeri tutkituissa ryhmissä ei ollut eroa normaali , ylipainoisia ja lihavia yksilöidenkäytetyt tekniikat . Kuitenkin saanti energia , tyydyttymättömiä rasvoja , ( n - 3 ) ​​monityydyttymättömiä rasvoja , ( n - 6 ) monityydyttymättömiä rasvoja ja liukoista kuitua oli merkittäviä assosiaatioitaulosteen bakteereihin . Lisäksi yhteistyö - kaksoset identtiset energiankulutuksesta oli useampia numeroita ja DGGE - profiilin moninaiset Bacteroides spp . kuin yhteistyössä kaksoset eri saantia. Lisäksi yhteistyö kaksoset , jotka nautitaansamoja määriä tyydyttyneitä rasvoja oli hyvin samanlainen DGGE - profiilit Bacteroides spp . , Kun taas yhteinen kaksoset samanlaisia ​​kulutus kuitua oli hyvin alhainen bifidobakteerien DGGE profiilin samankaltaisuus . Sen jälkeenvaikutusenergian saantiulosteen mikrobistonryhmä 16 lihavien henkilöiden arvioitiin aikana12 kuukautta intervention , joka koostui6 viikkoa vähän energiaa ruokavalio ( VLED ) ja sen jälkeenseuranta-aikana 5 , 8 ja 12 kuukautta. Ruokavalio suunnitelma yhdistettiin liikunnan ja elintapojen neuvonta . Ulosteen analysoitiin käyttäen qPCR , DGGE ja fluoresoiva in situ- hybridisaatio . Vaikutusvähäenergisen ruokavalioulosteen bakteerien lukumäärä kuvattiinlineaarisen yhdistettyä mallia , joka oli toistuvissa mittauksissa samaan yksilöön . VLED aikana vaikuttisuuri ulosteen päämikrobiryhmät , erityisesti bifidobakteerien laski lähtötasoon verrattuna numeroita . Lisäksimuutos numerotulosteen bakteeri tutkituissa ryhmissä , minkä jälkeenravinnosta eikäpainon muutoksia 12 kuukauden aikana . Metanogeenien havaittiin 56 % osallistujista jokaisella näytteenottokerralla pisteen , riippumattamuutoksen ravinnosta . Lisäksi laihtuminen liittyilasku Lactobacillus ryhmän bakteereja . Nämä havainnot vahvistavat, ettäruokavalio ja energinen saanti on tärkeä rooli modulaatioonulosteen mikrobiston . Lopuksimahdollisuudet hyödyntäätietoa ekspressiotasojen valitun stressiä geenien laadun arvioinnissa probioottien tuotteita arvioitiin . Tätä varten käänteiskopiointia qPCR kehitettiin tutkia-vioitu clpL1 ja clpL2 stressiä geenien Lactobacillus rhamnosus VTT E - 97800 solujen altistuksen jälkeen käsittely - stressiolosuhteita tai jäädyttää - kuivaus . Lämpökäsittelyt suoritettiin L. rhamnosus VTT E - 97800 laboratoriomittakaavassa , kun taas hapon avulla tehtiin sekä laboratorio -ja fermentoria mittakaavassa. RNA uutettiin tuoreisiin soluihin ja kylmäkuivattiin jauheet . clpL1 ja clpL2 transkriptien analysoitiin qPCR käyttämällä SYBR Green I clpL1 aiheutettiin L. rhamnosus VTT E - 97800 -soluja altistetaan 50 ° C: seen ja paljon vähäisemmässä määrin solut altistuvat 47 ° C. Ei induktio havaittiin clpL2 aikana joko happo-tai lämpökäsittely tahansasovellettavat ehdot. RNA erillään kylmäkuivattu jauheet onnistunut , vaikka useat yritettiin korkealaatuisia tuotteita . Nämä tulokset viittaavat siihen, että laadun kehittäminen indikaattorit probioottien tuotteiden erojen perusteellailmaus stressi geenien onhaastava tehtävä , sillä melko ankaria ehtoja ilmeisesti tarvitaan havaita erojageenien ilmentyminen . Lisäksihävinnyt RNA erillään Kuiva- pow - Ders haittaasovellettavuutta tätä tekniikkaalaadunvalvonta probioottien tuotteita