Monitoring the cell number of Lactococcus lactis subsp. cremoris FC in human feces by real-time PCR with strain-specific primers designed using the RAPD technique

Strain-specific PCR primers for Lactococcus lactis subsp. cremoris FC were developed using the randomly amplified polymorphic DNA (RAPD) technique. RAPD was used to generate strain-specific markers. A 1164-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific for L. lactis subsp. cremoris FC was designed. The specificity of this primer pair was tested with 23 L. lactis subsp. cremoris strains and 20 intestinal bacterial species, and was found to be strain-specific. Subsequently, this primer pair was subjected to the quantification of L. lactis subsp. cremoris FC in the feces of subjects fed fermented milk containing this strain. After administration, L. lactis subsp. cremoris FC was detected in the feces of all 7 subjects, with the maximum number being between 10(5) and 10(9) cells g(-1) of feces. Furthermore, this strain was detected in only one feces sample 2 weeks after administration was stopped. These results suggest that L. lactis subsp. cremoris FC can survive passage through the gastrointestinal tract.     

益生菌补充改善吡嗪酰胺致大鼠肝损伤及肠道菌群紊乱的效果

目的：吡嗪酰胺作为抗结核药物治疗结核病可引起患者肝损伤及肠道菌群紊乱,本研究拟探讨益生菌 （干酪乳杆菌（Lactobacillus casei,LcS））补充对吡嗪酰胺致大鼠肝损伤及肠道菌群紊乱的影响。方法：将40 只 成年雄性SD大鼠随机分为4 组,即正常对照组（NC组）、吡嗪酰胺组（L0组）、低剂量LcS组（L1组）、高剂 量LcS组（L2组）。L0组、L1组和L2组均给予吡嗪酰胺处理,以建立肝损伤模型;L0为阳性（药物肝损伤）组; L1组和L2组大鼠每天分别给予10、20 mL/（kg·d）LcS（108 CFU/mL）灌胃,持续10 周。苏木精-伊红染色观察 各组大鼠肝脏组织病理学变化;速率法检测各组大鼠血清丙氨酸氨基转移酶（alanine aminotransferase,ALT）和 天冬氨酸氨基转移酶（aspartate aminotransferase,AST）水平;实时荧光定量聚合酶链式反应技术对大鼠粪便中 的双歧杆菌、乳酸杆菌及大肠杆菌16S rDNA V3可变区进行定量分析。结果：经吡嗪酰胺处理10 周后,L0组大鼠 苏木精-伊红染色切片显示肝细胞中度水肿,出现明显的气球样变,肝索结构消失并伴有炎性细胞浸润,病理学 评分达到3.20 分,血清ALT及AST水平分别升高到95.90 U/L和188.60 U/L,显著高于正常对照组的73.90 U/L和 139.20 U/L,说明肝损伤造模成功。经过不同剂量的LcS干预10 周后,大鼠肝小叶结构均较L0组得到明显改善,病 理学评分、血清ALT和AST水平均降低到正常对照组水平。实验菌株定量分析结果显示,大鼠经过不同分组及干预 时间的延长,3 种代表菌株在组间及组内均具有统计学差异。从分组来看,高剂量LcS组大鼠在干预第6周末及干预 第10周末,双歧杆菌和乳酸杆菌的量均较L0组明显升高,大肠杆菌的量明显降低（P＜0.05）;从干预时间来看, 高剂量LcS组大鼠在干预第10周末,双歧杆菌和乳酸杆菌的量均较干预前显著增加,分别达到干预前的1.18 倍和 1.03 倍。结论：LcS对吡嗪酰胺诱导的肝损伤及肠道菌群紊乱具有一定的改善作用,且随着时间的延长及剂量的增 加,效果更明显。其作用机制可能与LcS维持肠道内环境稳态、调节肠道菌群有关。     

Use of steam condensing at subatmospheric pressures to reduce Escherichia coli O157:H7 numbers on bovine hide.

This study used a laboratory-scale apparatus to apply subatmospheric steam to bovine hide pieces inoculated with Escherichia coli O157:H7 in maximum recovery diluent (MRD) and in high-liquid content and low-liquid content fecal suspensions (HLC fecal and LLC fecal, respectively). The survival of the organism in fecal clods, which were stored for 24 days in a desiccated state, was assessed. Inoculated fecal clods were also treated with subatmospheric steam. Steam treatment at 80 +/- 2 degrees C for 20 s reduced E. coli O157:H7 concentrations on hide inoculated to initial concentrations of approximately 7 log10 CFU/g by 5.46 (MRD inoculum), 4.17 (HLC fecal inoculum), and 5.99 (LLC fecal inoculum) log10 CFU/g. The reductions achieved in samples inoculated with LLC feces were larger than in samples inoculated with HLC feces (P < 0.05). Treatment at 80 +/- 2 degrees C for 10 s resulted in significantly smaller reductions (P < 0.05) on hide pieces of 2.54 (MRD), 1.94 (HLC fecal), and 2.15 (LLC fecal) log10 CFU/g. There were no significant differences among the reductions observed in all inoculum types in samples treated for 10 s. E. coli O157:H7 inoculated in fecal clods to 7.78 log10 CFU/g and stored at 4 or 15 degrees C survived for at least 24 days. Steam treatment (20 s) of 3-day-old clods reduced surviving E. coli O157:H7 numbers from 4.20 log10 CFU/g to below the limit of detection of the assay used (1.20 log10 CFU/g). This study shows that steam condensing at or below 80 +/- 2 degrees C can reduce E. coli O157:H7 when present on bovine hide, reducing the risk of cross contamination to the carcass during slaughter and dressing.     

干酪乳杆菌LcS■发酵青稞植物饮料研究

研究了干酪乳杆菌LcS■在发酵青稞植物饮料中的生长动力学情况、pH变化和代谢组学分析,并对其进行了感官评价。结果表明：干酪乳杆菌LcS■发酵以青稞为主要原料的植物饮料延滞期为0～5 h,5～18 h活菌数从6.92 lg（CFU/mL）增加到9.52 lg（CFU/mL）,pH从5.40降至3.50。发酵完的青稞植物酸奶在色泽、风味口感和组织状态都具有较好的评分,口感酸甜适中,顺滑细腻。代谢组学分析显示,大豆皂苷、奎宁酸、多糖等具有抗肿瘤、增强免疫、抗氧化作用的功能性代谢产物在发酵前后的差异表达倍数值均＞1,发酵后表达量显著高于发酵前。综合考虑,干酪乳杆菌LcS■发酵的青稞植物饮料可作为一种功能性植物发酵饮品开发。     

Quantification of total viable bacteria on fish fillets by using ethidium bromide monoazide real-time polymerase chain reaction

Real-time PCR based on universal primers for amplification of a highly conserved bacterial 16S rDNA sequence was utilized in conjunction with the treatment of extracted bacterial cells with ethidium bromide monoazide (EMA) for the differential enumeration of viable and dead cells on cod fillets. Amplification of DNA from dead bacterial cells was successfully inhibited by EMA, whereas the DNA from viable cells was readily amplified. The detection range of the EMA real-time PCR assay was linear from 1 x 10(1) to 1 x 10(5) mixed bacterial genomic targets per PCR derived from broth cultures of fish tissue. The minimum detection limit of bacteria was found to be 1 x 10(1) genomic units/real-time PCR, equivalent to 1 x 10(5) CFU per gram of tissue. The EMA real-time PCR allowed construction of a standard curve obtained by plotting the log of genomic targets from strictly viable cells against resulting PCR cycles (Ct values) that facilitated quantification of total viable bacteria from fish fillets. The log of the total number of genomic DNA targets from EMA treated cells and plate counts from six randomly procured cod fillets were found not to be statistically different with the exception of one fillet. The process of freezing and thawing fillet tissue resulted in a drop in mean colony forming units (CFU) detected by plate counts from log 8.5+/-0.2 to log 8.1+/-0.1. A similar reduction in genomic targets from 8.5+/-0.1 to 8.0+/-0.16 was detected by EMA real-time PCR.     

Dietary supplementation with Lactobacillus- and Propionibacterium-based direct-fed microbials and prevalence of Escherichia coli O157 in beef feedlot cattle and on hides at harvest

The objective of this study was to describe the prevalence of Escherichia coli O157 in the feces and on the hides of finishing beef cattle fed a standard diet and those fed diets supplemented with direct-fed microbials. Two hundred forty steers received one of four treatments throughout the feeding period: (i) control: no added microbials; (ii) HNP51: high dose of Lactohacillius acidophilus strain NP 51 (10(9) CFU per steer daily) and Propionibacterium freudenreichii (10(9) CFU per steer daily); (iii) HNP51+45: high dose of NP 51 (10(9) CFU per steer daily), P. freudenreichii (10(9) CFU per steer daily), and L. acidophilus NP 45 (10(6) CFU per steer daily); or (iv) LNP51+45: low dose of NP 51 (10(6) CFU per steer daily), P. freudenreichii (10(9) CFU per steer daily), and NP 45 (10(6) CFU per steer daily). Samples were collected from each animal and analyzed for the presence of E. coli O157 using immunomagnetic separation methods on day 0 (feces), 7 days before harvest (feces), and at harvest (feces and hide). At the end of the feeding period, cattle receiving HNP51 were 57% less likely to shed detectable E. coli O157 in their feces than were the controls (P < 0.01). For animals receiving HNP51+45 and LNP51+45, fecal prevalence did not differ from that of the controls. The prevalence of positive hide samples was least among cattle receiving HNP51+45 (3.3%); these animals were 79% less likely (P < 0.06) to have a positive hide sample than were the controls (prevalence = 13.8%). There was poor agreement of the culture results between fecal and hide samples collected from the same animal (kappa = 0.08; confidence interval = -0.05 to 0.2). Cattle supplemented with a high dose of NP 51 had reduced E. coli O157 prevalence in both fecal and hide samples, indicating that this treatment may be an efficacious preharvest intervention strategy.     

Processing plant persistent strains of Listeria monocytogenes appear to have a lower virulence potential than clinical strains in selected virulence models

Listeria monocytogenes is an important foodborne bacterial pathogen that can colonize food processing equipment. One group of genetically similar L. monocytogenes strains (RAPD type 9) was recently shown to reside in several independent fish processing plants. Persistent strains are likely to contaminate food products, and it is important to determine their virulence potential to evaluate risk to consumers. We compared the behaviour of food processing persistent and clinical L. monocytogenes strains in four virulence models: Adhesion, invasion and intracellular growth was studied in an epithelial cell line, Caco-2; time to death in a nematode model, Caenorhabditis elegans and in a fruit fly model, Drosophila melanogaster and fecal shedding in a guinea pig model. All strains adhered to and grew in Caco-2 cells in similar levels. When exposed to 10(6) CFU/ml, two strains representing the persistent RAPD type 9 invaded Caco-2 cells in lower numbers (10(2)-10(3) CFU/ml) as compared to the four other strains (10(4)-10(6) CFU/ml), including food and human clinical strains. In the D. melanogaster model, the two RAPD type 9 strains were among the slowest to kill. Similarly, the time to reach 50% killed C. elegans worms was longer (110 h) for the RAPD type 9 strains than for the other four strains (80 h). The Scott A strain and one RAPD type 9 strain were suspended in whipping cream before being fed to guinea pigs and the persistent RAPD type 9 strain was isolated from feces in a lower level (approximately 10(2) CFU/g) than the Scott A strain (approximately 10(5) CFU/g) (P<0.05). The addition of NaCl has been shown to cause autoaggregation and increases adhesion of L. monocytogenes to plastic. However, growth in the presence of NaCl did not alter the behaviour of the tested L. monocytogenes strains in the virulence models. Overall, the two strains representing a very common fish processing plant persistent group (RAPD type 9) appear to have a lower virulence potential in all four virulence models than Scott A and a strain isolated from a clinical case of listeriosis.     

PCR detection of Bifidobacterium strains and Streptococcus thermophilus in feces of human subjects after oral bacteriotherapy and yogurt consumption

Streptococcus thermophilus, Bifidobacterium infantis Y1 and Bifidobacterium breve Y8 strains were identified and enumerated by PCR assay in human fecal samples after intake of the pharmaceutical preparation VSL-3 or yogurt. ThI/ThII primer set, specific for S. thermophilus, was selected testing its specificity against several strains of enterococci, streptococci and other genera colonizing the human intestine. A culture-independent PCR protocol, developed in this study, allowed to directly detect and enumerate S. thermophilus in human feces, excluding culture-based techniques or time consuming DNA isolation and purification procedures. Intestinal persistence of S. thermophilus was studied in feces of 10 healthy subjects given VSL-3 or yogurt. Streptococcal population was detected after 3 days of administration and persisted for 6 days after the treatment suspension. In the same trial, the colonization kinetics of B. infantis Y1 and B. breve Y8 were studied by amplification of colonies with the strain-specific primer sets InfY-BV.L/R and BreY-BV.R/L, showing a host-dependent transient colonization behaviour. PCR analysis of feces from 10 patients affected by inflammatory bowel diseases (IBD) and treated with VSL-3 for 2 months showed a colonization pattern of S. thermophilus, B. infantis Y1 and B. breve Y8 similar to that observed with the healthy subjects.     

Pathogenicity of enterohemorrhagic Escherichia coli in neonatal calves and evaluation of fecal shedding by treatment with probiotic Escherichia coli

The pathogenicity and fecal shedding of enterohemorrhagic Escherichia coli (EHEC) O26:H11, O111:NM, and O157:H7 were compared in calves (< 1 week of age) with or without prior treatment with probiotic bacteria (competitive exclusion E. coli). Three groups of 12 to 14 calves were used for these treatments. Half of the calves in each group were perorally administered 10(10) CFU of probiotic bacteria per calf, and, 2 days thereafter, 10(8) CFU of a five-strain mixture with one of the three EHEC serotypes per calf were administered to each calf. None of the EHEC serotypes caused clinical disease,and neither gross nor microscopic lesions attributable to EHEC were detected in control or probiotic-treated calves at necropsy. In calves administered E. coli O157:H7, fecal shedding was greatly reduced (> 6 log10 CFU/g) by 8 days after administration, and there was no significant difference (P > 0.05) in fecal shedding of E. coli O157:H7 between probiotic-treated and untreated control groups at that time. In contrast, control calves perorally administered E. coil of serotypes O111:NM or O26:H11 continued to shed substantial populations (10(2.1) to 10(6) CFU/g of feces and 10(2.5) to 10(4.9) CFU/g of feces, respectively) throughout 7 days postadministration of EHEC. In both groups administered either E. coli O111:NM or O26:H11, significantly less (P < 0.05) EHEC was isolated from feces at 7 days postadministration of EHEC and at necropsy from theprobiotic-treated group than from the untreated control group. Overall, neonatal calves shed in the feces from 1 to 7 days following peroral administration of EHEC greater populations of E. coli O111:NM and O26:H111 than E. coli O157:H7. In addition, treatment of calves with probiotic E. coli reduced fecal shedding of E. coli O111:NM and O26:H11 in most calves.     

Effect of bifidobacteria feeding on fecal flora and production of immunoglobulins in lactating mouse

To elucidate the effects of probiotics on the stimulation of immunoglobulin production during lactation, feeding trials of bifidobacteria in lactating mice were conducted. Bifidobacteria appeared in feces at 9.67+/-0.17 log 10 number per gram levels. All bifidobacteria found in the feces were the administered strain. Mice fed bifidobacteria for 12 days showed significantly high levels of fecal total IgA compared to that of the control group (P < 0.05). The levels of anti-beta-lactoglobulin IgA in milk as well as in fecal extracts were significantly higher in the bifidobacteria-fed group than that of the control group (P < 0.05). These results suggest that the intake of bifidobacteria can enhance local production of IgA in milk and the intestine, which may help to protect both pups and dams from exposure to food antigens.     

Recovery and metabolism of xanthohumol in germ‐free and human microbiota‐associated rats

The impact of human intestinal bacteria on the bioavailability of the prenylflavonoid xanthohumol (XN) was studied by comparing germ‐free (GF) and human microbiota‐associated (HMA) rats. After XN application, XN, XN conjugates, and isoxanthohumol (IX) conjugates occurred in blood samples of GF and HMA rats, whereas IX was detected only in the blood of HMA rats. Overall excretion of XN and its metabolites within 48 h was only 4.6% of the ingested dose in GF rats and 4.2% in HMA rats, feces being the major route of excretion. While both GF and HMA rats excreted XN, IX, and their conjugates with urine and feces, 8‐prenylnaringenin and its corresponding conjugates were exclusively observed in the feces of HMA rats. The microbial formation of 8‐prenylnaringenin was confirmed by incubation of XN and IX with human fecal slurries. The amount of conjugates excreted in urine and feces was lower in HMA rats compared to GF rats indicating their hydrolysis by human intestinal microbiota. Thus, the impact of bacteria on the XN metabolism in the gut may affect the in vivo effects of ingested XN.     

Starter culture activity in refrigerated fermented soymilk.

The refrigerated shelf life of soymilk fermented with single cultures of Lactobacillus fermentum, L. casei, Streptococcus salivarius subsp. thermophilus, and Bifidobacterium longum was evaluated. During storage at 4 degrees C for 28 days, the stability of the microflora differed markedly among the starter cultures. After 28 days, the average numbers of S. salivarius subsp. thermophilus decreased by two log cycles to 6.0 x 10(7) CFU/ml, whereas those of L. casei increased gradually by more than two log cycles to 4.6 x 10(9) CFU/ml. Numbers of B. longum and L. fermentum remained moderately high (8.7 x 10(8) CFU/ml and 3.7 x 10(8) CFU/ml, respectively) even after 28 days of storage. S. salivarius subsp. thermophilus and L. casei continued to metabolize sucrose during the storage period, but the pattern of consumption was different among the strains. The other starter cultures did not seem to have significant activity (P > 0.05) on the residual sugars. In most cases, L(+)-lactate predominated.     

Contamination flows of Bacillus cereus and spore-forming aerobic bacteria in a cooked,pasteurized and chilled zucchini purée processing line

A food processing plant producing pasteurized purées and its zucchini purée processing line were examined for contamination with aerobic and facultative anaerobic bacterial spores during a day's operation. Multiplication of spores was also monitored in the product stored under different conditions. High concentrations of Bacillus cereus spores were found in the soil in which the zucchinis were grown (4.6+/-0.3 log CFU/g), with a background spore population of 6.1+/-0.2 log CFU/g. In the processing plant, no B. cereus or psychrotrophic bacterial spores were detected on equipment. B. cereus and psychrotrophic bacterial spores were detected after enrichment in all samples of raw zucchinis, washed zucchinis, of two ingredients (starch and milk proteins) and in processed purée at each processing step. Steam cooking of raw zucchinis and pasteurization of purée in the final package significantly reduced spore numbers to 0.5+/-0.3 log CFU/g in the processed food. During storage, numbers of spore-forming bacteria increased up to 7.8+/-0.1 log CFU/g in purée after 5 days at 20-25 degrees C, 7.5+/-0.3 log CFU/g after 21 days at 10 degrees C and 3.8+/-1.1 log CFU/g after 21 days at 4 degrees C. B. cereus counts reached 6.4+/-0.5 log CFU/g at 20-25 degrees C, 4.6+/-1.9 log CFU/g at 10 degrees C, and remained below the detection threshold (1.7 log CFU/g) at 4 degrees C. Our findings indicate that raw vegetables and texturing agents such as milk proteins and starch, in spite of their low levels of contamination with bacterial spores and the heat treatments they undergo, may significantly contribute to the final contamination of cooked chilled foods. This contamination resulted in growth of B. cereus and psychrotrophic bacterial spores during storage of vegetable purée. Ways to eliminate such contamination in the processing line are discussed.     

Short communication: Examination of milk filters by real-time PCR as a herd-level indicator of the presence of Mycobacterium avium subspecies paratuberculosis in dairy herds

The aim of this study was to assess the suitability of real-time quantitative PCR (qPCR) for the detection of Mycobacterium avium ssp. paratuberculosis (MAP) in milk filters as a herd level indicator of paratuberculosis infection. Seventy-nine samples from textile or metal milk filters from 15 herds with defined MAP prevalence (infection status = noninfected, 0-5%, 5-10%, or >10% of animals with clinically confirmed paratuberculosis) were analyzed. The MAP DNA was isolated by a modified commercially available protocol for feces, and detection and quantification of the pathogen was performed by the IS900 qPCR. Mycobacterium avium ssp. paratuberculosis DNA was detected in 63 (79.7%) samples. Determination of MAP infection established by fecal and tissue culture was correctly confirmed by the analysis of milk filters on 11 of 12 infected farms; MAP was not detected in filters from 3 farms where paratuberculosis was never diagnosed. Statistical analysis of the data supports the evidence that milk filters can be used as a template for the direct detection of MAP on the herd level. The probability of successful MAP detection in milk filters in a herd with MAP-infected cows is at least 94.3%. Absolute numbers of MAP detected on the milk filter can be used for a rough estimation of paratuberculosis prevalence >10% in the herd. Analysis of milk filters for the presence of MAP can be a useful tool for the detection of paratuberculosis on the herd level before any individual control strategies.     

Development of an experimental model to assess the ability of Escherichia coli O157:H7-inoculated fecal pats to mimic a super shedder within a feedlot environment

This study was conducted to develop an experimental model that could assess the ability of Escherichia coli O157:H7-inoculated fecal pats to mimic a super shedder (>10(4) CFU/g of feces) within a feedlot environment. The day before the study began, 48 steers that had been negative for E. coli O157:H7 in feces for three consecutive weeks were sorted into three treatment groups, with two replicate pens per treatment and 8 steers per pen. Steers within the pens (20.50 by 10.75 m) were exposed to control feces or feces inoculated with two levels of a mixture of five strains of nalidixic acid-resistant E. coli O157:H7 (low level, 10(2) CFU/g; high level, 10(5) CFU/g). Five 300-g fecal pats were introduced into the pens twice daily (10:00 a.m. and 2:30 p.m.) on days 0 through 6 and days 14 through 20. Pats were placed in the pen at random locations to mimic defecation of a steer within the pen. Fecal grab samples, hide swab samples (500-cm2 area of the rump), natural fecal pat samples (freshly voided), and rope samples (1.22-m-long manila rope) where obtained at multiple times during the 49-day trial to evaluate the spread of nalidixic acid-resistant E. coli O157:H7 throughout the feedlot environment and among penmates. Immunomagnetic separation and selective media were used to detect E. coli O157:H7. Nalidixic acid-resistant E. coli O157:H7 was detected in 13 high-level treatment fecal grab samples, 7 high-level treatment hide swab samples, 1 low-level hide swab sample, and 2 high-level rope samples. For both fecal grab and hide swab samples, the overall prevalence of E. coli O157:H7 in the high-level group was greater (P < 0.01) than that for the pooled low-level and control groups. Addition of inoculated fecal pats to pens increased transmission of E. coli O157:H7 among penmates, but cattle that acquired E. coli O157:H7 shed the bacterium for only a short time at low levels. Transmission of E. coli O157:H7 from the feces of super shedders to naive penmates may contribute to the observed transient nature of shedding of E. coli O157:H7 among feedlot cattle.     

Probiotic effects of Lactobacillus casei on DSS-induced ulcerative colitis in mice

We tested the effect of Lactobacillus casei strain Shirota (LcS) on the murine model of ulcerative colitis induced by dextran sodium sulphate. The effect of LcS was tested either as a prophylactic 10 days before the onset of the disease, simultaneously with ulcerative colitis induction or continued 10 days after the disease was induced. LcS was not able to prevent the disease induction in any of the experiments. However, important clinical parameters including blood anemia indicators, body weight, and organ weight were improved in the animals receiving LcS as compared with the ulcerative colitis-induced controls. Increased colonic epithelial regeneration in the LcS treated animals was observed in the chronic stage. The results seemed better for the simultaneous short LcS treatment where some parameters remained similar to the PBS controls, including disease activity scores measured in the acute stage. We can conclude that although LcS alone cannot prevent the induction of ulcerative colitis by dextran sodium sulphate, it can improve the clinical condition of the mice. This could imply important biological consequences for the human situation. Further studies including LcS or other probiotic bacteria together with the available treatment are encouraged.     

Impact of consumption of probiotic lactobacilli-containing yogurt on microbial composition in human feces

An in vivo study was carried out to determine the effect of consuming probiotic lactobacilli-containing yogurt on the composition of microbiota in the human gut. Fifteen healthy adults ingested a daily serving of one of three commercial yogurts (two of the products contained a probiotic lactobacilli strain) for 20 days. Fecal samples at defined time points before, during, and after the period of yogurt ingestion were collected and analyzed. The fecal population of lactobacilli was determined by a culture-based method and subsequent colony PCR for the identification of species. Six predominant bacterial groups in the fecal samples were quantitatively determined based on a sequence-specific SSU rRNA cleavage method coupled with a suite of oligonucleotide probes, which was optimized for the target-specific detection of bacterial groups inhabiting human feces. In the ingestion period, one probiotic strain was detected in the feces of all five subjects who consumed the yogurt containing the strain, while the other strain was detected in three of another five subjects. The population levels of the two major groups (Bacteroides and Prevotella, and the Clostridium coccoides-Eubacterium rectale group) in the fecal samples tended to change in response to the ingestion but the change did not seem to be dependent on the product-specific property of each yogurt. These results suggest that the human fecal bacterial community could be altered by ingesting yogurt, although whether probiotic lactobacilli are present or absent in the yogurt does not seem to be a factor in this change.     

Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial cytochrome b targets

We designed and tested real-time PCR probe/primer sets to detect and quantify Cytochrome b sequences of mitochondrial DNA (mtDNA) from nine vertebrate species of pet (dog), farm (cow, chicken, sheep, horse, pig), wildlife (Canada goose, white-tailed deer), and human. Linear ranges of the assays were from 10(1) to 10(8) copies/microl. To formally test the performance of the assays, twenty blinded fecal suspension samples were analyzed by real-time PCR to identify the source of the feces. Sixteen of the twenty samples were correctly and unambiguously identified. Average sensitivity was calculated to be 0.850, while average specificity was found to be 0.994. One beef cow sample was not detected, but mtDNA from 11 other beef cattle of both sexes and varying physiological states was found in concentrations similar (3.45 x 10(7) copies/g) to thatfound in human feces (1.1 x 10(7) copies/g). Thus, environmental conditions and sample handling are probably important factors for successful detection of fecal mtDNA. When sewage samples were analyzed, only human mtDNA (7.2 x 10(4) copies/ 100 mL) was detected. With a detection threshold of 250 copies/reaction, an efficient concentration and purification method resulted in a final detection limit for human feces of 1.8 mg/ 100 mL water.     

Behavior of Trp-P-1 and its metabolites in rat excreta

To study of the behavior of Trp-P-1 and its metabolites in rat feces and urine, rats were orally administered with Trp-P-1 (750, 1,500 and 2,500 micrograms/rat), and excreted Trp-P-1 was analyzed using HPLC assay and bacterial mutagenicity assay. The extraction of Trp-P-1 from urine was performed by using the chloroform extraction method, and blue rayon was used for the extraction from feces. When Trp-P-1 was added to rat feces and urine, the recoveries of Trp-P-1 were 85.9 +/- 3.9% and 91.3 +/- 3.7%, respectively. The extracts of feces and urine from rats administered with Trp-P-1 were individually fractionated by thin layer chromatography on C18 gel. The major mutagenic zone corresponding to Trp-P-1 was found at Rf 0.09 in both extracts, while the feces extract gave two additional mutagenic zones at Rf 0.15 and 0.20. More than 97% of the fecal mutagenic activity was due to unchanged Trp-P-1. In rats administered with 750 micrograms of Trp-P-1, the amount of extracted Trp-P-1 and the number of His+ colonies induced by whole excreta were 81.6 +/- 7.1 micrograms (n = 6) and (432 +/- 77) x 10(4) for feces, and 28.7 +/- 4.9 micrograms and (171 +/- 28) x 10(4) for urine. The recoveries of Trp-P-1 in the feces and urine were 10.8 +/- 0.9% and 3.8 +/- 0.7% by HPLC analysis, and 11.1 +/- 2.0% and 4.4 +/- 0.7% by mutagenicity assay respectively. The results of the two assays seemed to show similar patterns of recovery.     

Comparative evaluation of yogurt and low-fat cheddar cheese as delivery media for probiotic Lactobacillus casei

This study used Lactobacillus casei 334e, an erythromycin-resistant derivative of ATCC 334, as a model to evaluate viability and acid resistance of probiotic L. casei in low-fat Cheddar cheese and yogurt. Cheese and yogurt were made by standard methods and the probiotic L. casei adjunct was added at approximately 10(7) CFU/g with the starter cultures. Low-fat cheese and yogurt samples were stored at 8 and 2 degrees C, respectively, and numbers of the L. casei adjunct were periodically determined by plating on MRS agar that contained 5 microg/mL of erythromycin. L. casei 334e counts in cheese and yogurt remained at 10(7) CFU/g over 3 mo and 3 wk, respectively, indicating good survival in both products. Acid challenge studies in 8.7 mM phosphoric acid (pH 2) at 37 degrees C showed numbers of L. casei 334e in yogurt dropped from 10(7) CFU/g to less than 10(1) CFU/g after 30 min, while counts in cheese samples dropped from 10(7) CFU/g to about 10(5) after 30 min, and remained near 10(4) CFU/g after 120 min. As a whole, these data showed that low-fat Cheddar cheese is a viable delivery food for probiotic L. casei because it allowed for good survival during storage and helped protect cells against the very low pH that will be encountered during stomach transit.