The role of probiotics and synbiotics on treatment of gestational diabetes: systematic review and meta-analysis.

OBJECTIVE: This review investigated the efficacy of probiotics and/or synbiotics in gestational diabetes mellitus treatment by targeting insulin resistance, lipid metabolism, and anti-inflammatory effects in an updated trial. DATA SOURCES: The literature review was performed using the key words "Probiotics," "Synbiotics," and "Gestational Diabetes" in several databases, including PubMed, ScienceDirect, and the Cochrane Central Register of Controlled Trials. STUDY ELIGIBILITY CRITERIA: Eligible publication was screened independently by 2 reviewers. Studies included provided at least 1 of the following outcomes: (1) blood glucose marker, including fasting blood glucose level, fasting serum insulin level, and homeostasis model assessment insulin resistance; (2) blood lipid profiles, including triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol; and (3) nitric oxide and C-reactive protein. METHODS: All studies were reviewed using the critical appraisal Cochrane risk-of-bias tool for randomized trials. The descriptions of the extracted data were guided by the Preferred Reporting Items for Systematic Reviews 2020 statement with the Grading of Recommendations Assessment, Development, and Evaluation approach. This study was registered on the International Prospective Register of Systematic Reviews database (identification number: CRD42022375665). RESULTS: From 13 randomized controlled trials involving 896 patients, individuals with probiotic had significant reduction on homeostasis model assessment insulin resistance (mean difference, -0.72; 95% confidence interval, -1.07 to -0.38; I2, 96%; P=.00), fasting blood glucose level (mean difference, -3.79; 95% confidence interval, -6.24 to -1.34; I2, 93%; P=.00), and insulin level (mean difference, -2.43 mg/dL; 95% confidence interval, -3.37 to -1.48; I2, 54%; P=.00). Meanwhile for profile lipid, significant reduction of the mean difference was observed in the triglyceride (mean difference, -17.73 mg/dL; 95% confidence interval, -29.55 to - 5.9; P=.003) and C-reactive protein (mean difference, -1.93 dL; 95% confidence interval, -2.3 to -1.56; P=.00). CONCLUSION: Probiotic and synbiotic supplementations reduced the risk of insulin resistance and improved glycemic control, blood lipid profiles, and inflammation in women with gestational diabetes mellitus. Probiotics may be a viable option for gestational diabetes mellitus treatment; however, large-scale, well-designed randomized controlled trials with longer follow-up periods are required before they can be recommended to patients.

Early detection of a possible multidrug-resistant Acinetobacter baumannii outbreak in the local hospital setting by using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR), oxacillinase gene profiles, and antibiograms.

Background and Objectives: Detecting the source of a potential outbreak of multidrug resistant (MDR) Acinetobacter baumannii is necessary to be investigated. This study aimed to detect the possibility of A. baumannii outbreak in a hospital setting using a combination of random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR), antibiograms, and the presence of oxacillinase genes. Materials and Methods: The antibiogram of 31 clinical isolates and six environmental isolates of A. baumannii were determined by Vitek® 2 Compact. Oxacillinase genes (OXA-23, -24, -51, and -58) were detected by PCR, and RAPD-PCR was conducted using DAF-4 and ERIC-2 primers. The Similarity Index and dendrogram were generated using GelJ v2.3 software. Results: The antibiograms showed that all MDR A. baumannii isolates has very limited susceptibility to cephalosporins, but mostly susceptible to tigecycline. All isolates were positive for bla OXA-51-like gene, thirty-two of 37 total isolates (86.5%) were positive for bla OXA-23-like gene, and none were positive for bla OXA-24-like and bla OXA-58-like genes. RAPD-PCR showed that the DAF-4 primer on average had more band visualization and lower Similarity Index's variation compared to the ERIC-2. The discriminatory power of DAF-4 was 0.906. There was a significant correlation between the DAF-4 dendrogram pattern with the antibiogram (r=0.494, p<0.001) and the presence of bla OXA-23-like gene (r=0.634, p<0.001) from all ICU A isolates. Six out of fourteen ICU A isolates belonged to the same cluster with >95% Similarity Index, while one clinical isolate having an identical dendrogram and antibiogram pattern with an environmental isolate within this cluster. Conclusion: There is a high probability of MDR A. baumannii outbreak within ICU A detected by multiple analysis of RAPD-PCR, antibiogram and the bla OXA-23-like gene profiles. This combinatorial approach is conceivable to mitigate possible outbreak situations of A. baumannii in the local hospital without sophisticated microbiology laboratory.

Weissella confusa F213 ameliorated inflammation and maintained intestinal mucosa integrity in chemically induced colitis rats.

OBJECTIVE: This study was performed to investigate the potential effects of Weissella confusa F213 (WCF213) on chemically-induced colitis rats. Twelve male Wistar rats were divided into three groups: T1 (saline sterile), T2 (2.5% dextran sulfate sodium (DSS)- for 7 days), and T3 (WCF213 for 14 days, continued with 2.5% DSS for 7 days). The disease activity index (DAI) was monitored. After sacrificing the rats, the colon was collected for length measurement, local TNF-α level, HE staining for histology, and ZO-1 expression by using immunohistochemistry. RESULTS: WCF213 administration prevented weight loss and haematochezia, maintained average colon length and alleviated the clinical symptom of colitis, such as diarrhoea, albeit statistically non-significant (p < 0.05) compared with the T2 group. The histopathology of WCF213-treated colitis rats showed better architecture and less inflammatory cell infiltration into colon tissue. WCF213 significantly maintained the expression of ZO-1 in the mucosa (p < 0.001) and markedly reduced mucosal TNF-α concentration (p < 0.001) compared with the DSS group. Hence, these findings suggested that WCF213 attenuated clinical symptoms and inflammation and maintained mucosal integrity in DSS-induced colitis in vivo.

Enhancement of intestinal epithelial barrier function by Weissella confusa F213 and Lactobacillus rhamnosus FBB81 probiotic candidates in an in vitro model of hydrogen peroxide-induced inflammatory bowel disease.

OBJECTIVE: Weissella confusa F213 (WCF213) and Lactobacillus rhamnosus FBB81 (LrFBB81) are two probiotic candidates isolated from humans in our previous study. Their functional activity on the mucosal barrier has not yet been adequately investigated. Therefore, the objective of this study was to investigate the effect of these strains on maintaining mucosal integrity in vitro. Caco-2 cell monolayers were pretreated with WCF213 and LrFBB81 before being exposed to hydrogen peroxide. The integrity of mucosal cells was evaluated by measuring the transepithelial resistance (TER), flux of FITC-labelled dextran, and ZO-1 protein distribution with the help of an immunofluorescence method. RESULTS: WCF213 was found to significantly maintain the TER better than the control hydrogen peroxide-treated cells (p < 0.001), followed by the strain combination, and LrFBB81 alone (p < 0.05). The permeability of mucosa was also successfully maintained by the WCF213 strain. This was illustrated by the significant reduction in the flux of FITC-labelled dextran (p < 0.05), which was larger than that exhibited by the other groups. The ZO-1 distribution of strain-treated cells showed less disruption than hydrogen peroxide-treated cells, consistent with the TER and FITC experimental results. These findings indicate that WCF213 and LrFBB81 plays important roles in the maintenance of mucosal integrity in a strain-dependent manner.

Gut microbiota profile in healthy Indonesians.

BACKGROUND: Recently, gut microbiota has been associated with various diseases other than intestinal disease. Thus, there has been rapid growth in the study of gut microbiota. Considering the numerous factors influencing gut microbiota such as age, diet, etc., area-based research is required. Indonesia has numerous different tribes and each of these tribes have different lifestyles. Hence, it is expected that each tribe has a specific gut microbiota. A deeper insight into the composition of gut microbiota can be used to determine the condition of gut microbiota in Indonesians and to consider which treatment may be suitable and effective to improve health status. AIM: To investigate the gut microbiota of Indonesian subjects represented by Javanese and Balinese tribes by analyzing fecal samples. METHODS: Fecal samples were collected from a total of 80 individuals with 20 in each of the young groups ranging from 25-45 years and the elderly group aged 70 years or more from two different regions, Yogyakarta and Bali. Fecal sample collection was performed at the end of the assessment period (day 14 ± 1 d) during which time the subjects were not allowed to consume probiotic or antibiotic products. The quantification of various Clostridium subgroups, Lactobacillus subgroups, Enterococcus, Streptococcus, Staphylococcus, Bacteroides fragilis group and Prevotella, Bifidobacterium and Atopobium cluster, Enterobacteriaceae and Pseudomonas was performed using the Yakult intestinal flora-scan (YIF-SCAN). RESULTS: The bacterial population in younger subjects' feces was higher than that in the elderly population, with a total of approximately 10.0 - 10.6 log10 bacterial cells/g feces. The most abundant bacteria in all groups were Clostridium, followed by Prevotella, Atopobium, Bifidobacterium and Bacteroides. In the elderly, an increase in Enterobacteriaceae, Coliform and Escherichia coli was found. In terms of bacterial counts in Yogyakarta, total bacteria, Clostridium coccoides (C. coccoides) group, Bifidobacterium, Prevotella, Lactobacillus plantarum subgroup, and Streptococcus were significantly higher (P < 0.05) in younger than elderly subjects, while the Lactobacillus gasseri subgroup, Lactobacillus casei subgroup, and Lactobacillus reuteri subgroup counts were significantly lower (P < 0.05) in younger subjects. In Balinese subjects, total bacteria, C. coccoides group, Clostridium leptum subgroup, Bacteroides fragilis group, and Prevotella were significantly higher (P < 0.05) in younger compared to elderly individuals, while the Lactobacillus ruminis subgroup, and Enterobacteriaceae were significantly lower (P < 0.05) in younger subjects. The results also revealed that, besides the C. coccoides group and Clostridium leptum group being the most abundant gut microbiota in both Yogyakarta and Balinese people, the latter was indicated by a higher Clostridium perfringens count, which was almost 10 times that of Yogyakarta subjects. This may be a response to different lifestyles in the different tribes; however, this phenomenon requires further extensive study. CONCLUSION: Bacterial populations were higher in younger than in elderly subjects. Most abundant bacterial groups were Clostridium, Prevotella, Atopobium, Bifidobacterium, and Bacteroides. The level of Clostridium perfringens in Yogyakarta subjects was lower than that in Balinese subjects.

Author Correction: Diversity in gut bacterial community of school-age children in Asia.

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Anchorless cell surface proteins function as laminin-binding adhesins in Lactobacillus rhamnosus FSMM22.

Anchorless cell surface proteins (CSPs) were extracted with 1 M lithium chloride solution from Lactobacillus rhamnosus FSMM22. Loss of the anchorless CSPs resulted in a 2-fold decrease in FSMM22 cells bound to a constitutive extracellular matrix glycoprotein, laminin, in vitro. DNA-binding protein HU, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and 30S ribosomal protein S19 (RpsS) were identified by mass spectrometry in the extract as laminin-binding adhesins. Among the four proteins, RpsS was immunohistochemically confirmed to exist on the cell surface. Our findings strongly suggest that anchorless CSPs can enhance bacterial adhesion to the host.

In vitro safety assessments and antimicrobial activities of Lactobacillus rhamnosus strains isolated from a fermented mare's milk.

Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4-8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α-chymotrypsin and ß-glucosidase were detected, but those of α-galactosidase, ß-glucuronidase and N-acetyl-ß-glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell-free culture supernatant inhibited growths of Salmonella Typhimurium LT-2, Shigella sonnei, Listeria monocytogenes, and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain.

Effects of prebiotic oligosaccharides consumption on the growth and expression profile of cell surface-associated proteins of a potential probiotic Lactobacillus rhamnosus FSMM15.

To investigate carbohydrate preference of a potential probiotic, Lactobacillus rhamnosus FSMM15, six prebiotics, including two milk-derived prebiotics, galactooligosaccharides and lacto-N-biose I, and four plant-origin prebiotics, beet oligosaccharide syrup, difructose anhydride III, fructooligosaccharides, and raffinose, were examined. The strain utilized the milk-derived prebiotics at similar levels to glucose but did not utilize the plant-origin ones in the same manner, reflecting their genetic background, which allows them to adapt to dairy ecological niches. These prebiotics had little influence on the expression pattern of cell surface-associated proteins in the strain; however, an ATP-binding cassette transporter substrate-binding protein and a glyceraldehyde-3-phosphate dehydrogenase were suggested to be upregulated in response to carbon starvation stress.

Diversity in gut bacterial community of school-age children in Asia.

Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet.

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

Biodesulfurization of dibenzothiophene and its derivatives using resting and immobilized cells of Sphingomonas subarctica T7b.

The desulfurization ability of Sphingomonas subarctica T7b was evaluated using resting and immobilized cells with dibenzothiophene (DBT), alkyl DBTs, and commercial light gas oil (LGO) as the substrates. The resting cells of S. subarctica T7b degraded 239.2 mg of the initial 250 mg of DBT/l (1.36 mM) within 24 h at 27 degrees C, while 127.5 mg of 2-hydroxybiphenyl (2-HBP)/l (0.75 mM) was formed, representing a 55% conversion of the DBT. The DBT desulfurization activity was significantly affected by the aqueous-to-oil phase ratio. In addition, the resting cells of S. subarctica T7b were able to desulfurize alkyl DBTs with long alkyl chains, although the desulfurization rate decreased with an increase in the total carbon number of the alkylated DBTs. LGO with a total sulfur content of 280 mg/l was desulfurized to 152 mg/l after 24 h of reaction. Cells immobilized by entrapment with polyvinyl alcohol (PVA) exhibited a high DBT desulfurization activity, including repeated use for more than 8 batch cycles without loss of biodesulfurization activity. The stability of the immobilized cells was better than that of the resting cells at different initial pHs, higher temperatures, and for DBT biodesulfurization in successive degradation cycles. The immobilized cells were also easily separated from the oil and water phases, giving this method great potential for oil biodesulfurization.

Isolation of potential probiotic Lactobacillus rhamnosus strains from traditional fermented mare milk produced in Sumbawa Island of Indonesia.

To explore potential probiotics in the traditional foods of Indonesia, fermented mare milk produced in Sumbawa Island was investigated in this study. Gram stain, catalase activity, gas production, cell morphology, carbohydrate utilization pattern, and 16S rDNA sequencing were performed to identify isolated lactic acid bacteria. To assess their probiotic ability, tolerance of low pH, bile salts, artificial gastrointestinal fluids, and adhesion properties to extracellular matrices, were examined. In total 27 strains, 25 Lactobacillus rhamnosus and two Lactobacillus fermentum, were obtained. Among the isolated lactobacilli, three Lb. rhamnosus strains, FSMM15, FSMM22, and FSMM26, were selected as candidates for probiotics, using Lb. rhamnosus GG as index. In vitro binding assay of the three strains against several extracellular matrix proteins revealed that FSMM15 and FSMM26 gave greater binding ratios of mucin/bovine serum albumin (BSA) and significantly higher adhesive abilities to fibronectin than Lb. rhamnosus GG. FSMM22 showed significantly higher adhesion to laminin than Lb. rhamnosus GG.

Molecular characterization of the relationships among Amylomyces rouxii, Rhizopus oryzae, and Rhizopus delemar.

Twenty-one strains of Amylomyces rouxii isolated from starters of Asian fermented foods were divided into two groups, lactic acid (LA) and fumaric and malic acid (FMA) producers, by organic acid productivity in liquid culture. Phylogenetic analysis based on the ldhB gene, ribosomal RNA encoding DNA (rDNA) internal transcribed spacer (ITS) sequence, and genome-wide amplified fragment length polymorphism (AFLP) revealed that A. rouxii was grouped into two major clusters as to organic acid accumulation, corresponding to Rhizopus oryzae and Rhizopus delemar. These observations suggest that the species A. rouxii is composed of two distinct types, derived from R. oryzae or R. delemar via domestication in the starters.

Effects of long-term ingestion of difructose anhydride III (DFA III) on intestinal bacteria and bile acid metabolism in humans.

Changes in the intestinal microbiota of 10 human subjects with long-term ingestion of 3 g/d difructose anhydride III (DFA III; 4 persons, 2 months; 3 persons, 6 months; and 3 persons, 12 months) were examined by denaturing gradient gel electrophoresis (DGGE). According to the answers to questionnaires, the subjects were divided into two groups (constipated and normal). The DGGE profile was different for every individual and each subject had unique profiles of intestinal microbiota. In the DGGE profiles of constipated subjects, the intensities of bands related to Bacteroides spp. increased. Moreover, the DFA III-assimilating bacteria, Ruminococcus sp. were isolated from subjects who ingested DFA III for 12 months. These strains showed 95% similarity of their 16S rDNA sequences with that of Ruminococcus obeum ATCC 29174(T) (X85101) and produced large amounts of acetic acid. DFA III ingestion for 2 months tended to increase total organic acids in feces, and tended to decrease fecal pH and the secondary bile acid (SBA) ratio in total bile acids. The SBA ratio in total bile acids corresponded to fecal pH. The production of SBA was decreased by low pH in vitro. These results indicated that DFA III ingestion in humans tended to lower intestinal pH, inhibited bile acid 7alpha-dehydroxylation activities and also tended to decrease the SBA ratios in total bile acids. Moreover, as another cause for the decrease in the SBA ratio in total bile acids, it was suggested that the number of bile acid 7alpha-dehydroxylating bacteria were decreased by DFA III ingestion.

Effects of difructose anhydride III (DFA III) administration on rat intestinal microbiota.

The effects of difructose anhydride III (di-D-fructofuranose-1,2':2,3'-dianhydride; DFA III) administration (3% DFA III for 4 weeks) on rat intestinal microbiota were examined using denaturing gradient gel electrophoresis (DGGE). According to DGGE profiles, the number of bacteria related to Bacteroides acidofaciens and uncultured bacteria within the Clostridium lituseburense group decreased, while that of bacteria related to Bacteroides vulgatus, Bacteroides uniformis and Ruminococcus productus increased in DFA III-fed rat cecum. In the cecal contents of DFA III-fed rats, a lowering of pH and an increase in short chain fatty acids (SCFAs), especially acetic acid, were observed. The DFA III-assimilating bacterium, Ruminococcus sp. M-1, was isolated from the cecal contents of DFA III-fed rats. The strain had 98% similarity with R. productus ATCC 27340T (L76595), and mainly produced acetic acid. These results confirmed that the bacteria harmful to host health were not increased by DFA III administration. Moreover, DFA III stimulated the growth of Ruminococcus sp. M-1 producing acetic acid, which may alter the intestinal microbiota towards a healthier composition. It is expected that DFA III would be a new candidate as a prebiotic.

Effects of difructose anhydride III (DFA III) administration on bile acids and growth of DFA III-assimilating bacterium Ruminococcus productus on rat intestine.

The growth of DFA III-assimilating bacteria in the intestines of rats fed 3% DFA III for 2 weeks was examined. Sixty-four percent of the DFA III intake had been assimilated on day 3 of ingestion, and almost all of the DFA III was assimilated at the end of the experiment. The DFA III-assimilating bacterium, Ruminococcus productus, in DFA III-fed rats was in the stationary state of 10(8)-10(9) cells/g dry feces within a week from 10(6) cells/g dry feces on day 1 of DFA III ingestion. The number of R. productus cells was associated with the amount of DFA III excreted in the feces. The acetic acid produced from DFA III by R. productus lowered the cecal pH to 5.8. In control-fed rats and DFA III-fed rats, 94% of secondary bile acids and 94% of primary bile acids, respectively, were accounted for in the total bile acids analyzed. DFA III ingestion increased the ratio of primary bile acids and changed the composition of fecal bile acids. In conclusion, R. productus assimilated DFA III, produced short chain fatty acids, and the cecal pH was lowered. The acidification of rat intestine perhaps inhibited secondary bile acid formation and decreased the ratio of secondary bile acids. Therefore, it is expected that DFA III may prevent colorectal cancer and be a new prebiotic candidate.

Effects of indigenous starter cultures on the microbial and physicochemical characteristics of Urutan, a Balinese fermented sausage.

Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25 degrees C at night to 50 degrees C during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P. acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.

The effects of di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III) administration on human intestinal microbiota.

Di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III) was shown to enhance Ca absorption in rat and human intestine. The effects of DFA III administration (9 g per day for 4 weeks that corresponded to 3-fold the optimal dosage of DFA III) on human intestinal microbiota were studied using denaturing gradient gel electrophoresis (DGGE). The major groups of human intestinal microbiota reported previously: the Bacteroides, the Clostridium coccoides group (Clostridium cluster XIVa), the Clostridium leptum group (Clostridium cluster IV), and the Bifidobacterium group were detected. The similarity of 30 DGGE profiles based on the V3 region (before and after administration to the 15 subjects) of the 16S rDNA were calculated using Pearson's correlation based on numbers, positions and intensity of bands, and then a dendrogram of DGGE profiles was constructed by the unweighted pair group method using arithmetic average (UPGMA) clustering method. By these analyses, no difference in DGGE profiles after DFA III administration was observed in healthy subjects, while two subjects with chronic constipation showed different profiles, namely on numbers, positions and the intensity of some bands. Their stools were softer and stool frequencies increased and they obtained relief from constipation.

rDNA ITS sequence of Rhizopus oryzae: its application to classification and identification of lactic acid producers.

Rhizopus oryzae is an important organism for its production of organic acids such as lactic acid, fumaric acid, etc. To date, there were no easy methods to classify strains according to their acid production. The sequences of the ribosomal RNA-encoding DNA (rDNA) internal transcribed spacer (ITS) region of 64 strains of R. oryzae were analyzed and found to conserve mutations correspond to acid production. We have devised a way to use these mutations for a novel method to identify lactic-acid-producing Rhizopus oryzae, by designing specific polymerase chain reaction (PCR) primers on them. Touch down PCR using these primers amplified the ITS DNA of lactic acid producers specifically. By this method, we could isolate lactic acid producing strains from Indonesian fermented foods.