Two new Tytthoscincus Linkem, Diesmos, Brown (Squamata; Scincidae) from Peninsular Malaysia and another case of microsyntopy between ecologically specialized, unrelated, leaf-litter species.

Two new species of diminutive, sympatric, lowland, leaf-litter skinks of the genus Tytthoscincus Linkem, Diesmos Brown from the Sekayu region of Hulu Terengganu, Terengganu State in northeastern Peninsular Malaysia are described on the basis genetic and morphological data. One of the new species, T. monticolus sp. nov., was collected in a hilly riparian area along Sungai (=river) Bubu and is most closely related to an undescribed species from the Tembak Reservoir area. The other, T. keciktuek sp. nov. collected along Sungai Peres, is most closely related to T. perhentianensis Grismer, Wood, Grismer from Pulau (=island) Perhentian Besar. Sympatry and syntopy of multiple, specialized, unrelated, leaf-litter species of Tytthoscincus was previously only known from upland areas and these new species represent the first example of lowland of sympatry. More importantly, however, these endemic species add to a growing body of research and discoveries that continue to underscore the unrealized biodiversity of the riparian systems of Hulu Terengganu and the Sekayu region and their need for protection and continued study.

Estimation of uncertainty from method validation data: Application to a reverse-phase high-performance liquid chromatography method for the determination of amino acids in gelatin using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent.

A detailed procedure for estimating uncertainty according to the Laboratory of Government Chemists/Valid Analytical Measurement (LGC/VAM) protocol for determination of 18 amino acids in gelatin is proposed. The expanded uncertainty was estimated using mainly the method validation data (precision and trueness). Other sources of uncertainties were contributed by components in standard preparation measurements. The method scope covered a single matrix (gelatin) under a wide range of analyte concentrations. The uncertainty of method precision, µ(P) was 0.0237-0.1128pmolµl(-1) in which hydroxyproline and histidine represented the lowest and highest values of uncertainties, respectively. Proline and phenylalanine represented the lowest and highest uncertainties value for method recovery, µ(R) that was estimated within 0.0064-0.0995pmolµl(-1). The uncertainties from other sources, µ(Std) were 0.0325, 0.0428 and 0.0413pmolµl(-1) that were contributed by hydroxyproline, other amino acids and cystine, respectively. Hydroxyproline and phenylalanine represented the lowest and highest values of expanded uncertainty, U(y) that were determined at 0.0949 and 0.2473pmolµl(-1), respectively. The data were accurately defined and fulfill the technical requirements of ISO 17025:2005.

Immunomodulatory effects of damnacanthal isolated from roots of Morinda elliptica.

Morinda elliptica Ridley (Rubiaceae) has been used traditionally as a medicine to treat various diseases in Malaysia and southeast Asia. In the present study we investigated the immunomodulatory effects of damnacanthal isolated from the roots of Morinda elliptica. The immunomodulatory effect of this compound was evaluated by using the lymphocyte proliferation assay with mouse thymocytes and human peripheral blood mononuclear cells (PBMC). In addition, the effect of the compound on PBMC cell cycle progression was studied by using flow cytometry. The production of human interleukin-2 and human inteleukin-12 cytokines was also assessed using the enzyme linked immunosorbent assay (ELISA) technique. The lymphocyte proliferation assay showed that damnacanthal was able to activate mouse thymocytes and PBMC at a low concentration (0.468 microg/mL). Moreover, the production of human interleukin-2 and human interleukin-12 cytokines in the culture supernatant from damnacanthal activated lymphocytes was markedly up-regulated at 24 h and sustained until 72 h with a slight decrease with time. A positive correlation was found between the level of these two cytokines and the MTT-based proliferation assay. Based on the above results, damnacanthal can act as an immunomodulatory agent which may be very useful for maintaining a healthy immune system.

The impact of the level of the intestinal short chain Fatty acids in inflammatory bowel disease patients versus healthy subjects.

The aim of this study was to determine the changes of short chain fatty acids (SCFAs) in faeces of inflammatory bowel disease (IBD) patients compared to healthy subjects. SCFAs such as pyruvic, lactic, formic, acetic, propionic, isobutyric and butyric acids were analyzed by using high performance liquid chromatography (HPLC). This study showed that the level of acetic, 162.0 micromol/g wet faeces, butyric, 86.9 micromol/g wet faeces, and propionic acids, 65.6 micromol/g wet faeces, decreased remarkably in IBD faecal samples when compared with that of healthy individuals, 209.7, 176.0, and 93.3 micromol/g wet faeces respectively. On the contrary, lactic and pyruvic acids showed higher levels in faecal samples of IBD than in healthy subjects. In the context of butyric acid level, this study also found that the molar ratio of butyric acid was higher than propionic acid in both faecal samples. This might be due to the high intake of starch from rice among Malaysian population. It was concluded that the level of SCFAs differ remarkably between faecal samples in healthy subjects and that in IBD patients providing evidence that SCFAs more likely play an important role in the pathogenesis of IBD.

Gastrointestinal tract morphological alteration by unpleasant physical treatment and modulating role of Lactobacillus in broilers.

1. An experiment was conducted to determine the effects of supposedly unpleasant physical treatment on broiler performance, small intestinal development and ameliorating role of probiotics. 2. The following treatments were applied from day one: (1) chicks exposed to normal human contact fed basal diet (control); (2) chicks were exposed to unpleasant physical treatment and fed basal diet (UPT-BD); and (3) chicks were exposed to unpleasant physical treatment and fed basal diet supplemented with Lactobacillus (UPT-BDL). Chicks were exposed to UPT from days 1 to 21. Different segments of gastrointestinal tract were sampled at 14, 28, 35 and 42 d of age. 3. Broilers of UPT-BD had lower feed consumption compared with control group at 7 d of age. Overall, UPT-BDL birds showed higher body weight gain (BWG) and better feed conversion ratio (FCR) over the course of the experiment. 4. Birds of UPT-BD had lower concentrations of lactic, propionic and butyric acids in the caecum as compared with other groups at 14 d of age. Acetic acid concentration was profoundly decreased in both UPT groups compared to the control. 5. Duodenal villus height of UPT-BD broilers showed a slight reduction compared to the control and UPT-BDL birds at 14 d of age. Afterwards until day 42, UPT-BDL birds showed the highest villus height among treatments in different parts of the small intestine. 6. The results suggested that, even though UPT did not have significant inhibitory effects on the development of the small intestine and broiler performance, it negatively affected bacterial metabolic end products in the caecum, which could be ameliorated by the addition of Lactobacillus.

Selected microbial groups and short-chain fatty acids profile in a simulated chicken cecum supplemented with two strains of Lactobacillus.

Among the bacterial fermentation end products in the chicken cecum, butyrate is of particular importance because of its nutritional properties for the epithelial cell and pathogen inhibitory effects in the gut. An in vitro experiment, operated with batch bioreactor, was conducted to quantify butyric-producing bacteria in a simulated broiler cecum supplemented with Lactobacillus salivarius ssp. salicinius JCM 1230 and Lactobacillus agilis JCM 1048 during 24 h of incubation. Selected bacterial species were determined by real-time PCR and short-chain fatty acids and lactate concentrations were monitored. The results showed that after 24 h of incubation, Lactobacillus supplementation significantly increased the number of lactobacilli, bifidobacteria and Faecalibacterium prausnitzii in medium containing cecal content and lactobacilli supplementation (Cc + L) compared with the control (Cc). Addition of lactobacilli did not alter Escherichia coli and Clostridium butyricum, whereas it significantly (P < 0.05) reduced Salmonella in treatment Cc + L compared with the Cc treatment. Propionate and butyrate formation were significantly (P < 0.05) increased in treatment Cc + L as compared with the Cc treatment. Lactate was only detected in treatment containing 2 Lactobacillus strains. After 24 h of incubation, acetate concentration significantly (P < 0.05) decreased in all treatments. It was suggested that lactate produced by Lactobacillus in the cecal content improved the growth of butyric producers such as F. prausnitzii, which significantly increased butyrate accumulation. Additionally, the results showed that butyrate and propionate inhibited Salmonella without influencing the E. coli profile.

Growth of probiotic bacteria in trypticase phytone yeast medium supplemented with crude polysaccharides from Ganoderma lucidum

Ganoderma lucidum is a fungus usually used in traditional Chinese medicine. The high value of G. lucidum is related to its polysaccharides content. Crude polysaccharides from G. lucidum (GLCP) were obtained using hot water extraction method. There is about 0.57 g of GLCP in 1 g crude of G. lucidum. The prebiotic potential of GLCP was tested against probiotic bacteria namely: Bifidobacterium longum BB536, Bifidobacterium pseudocatenulatum G4, Lactobacillus acidophilus and Lactobacillus casei Shirota. The prebiotic potentials were studied in 10 mL basal Trypticase Phytone Yeast (abbreviated as bTPY) medium (without glucose) supplemented with various concentrations of GLCP (abbreviated as bTPYglcp) (0.5%, 1.0%, 1.5% and 2.0%). bTPY medium supplemented with glucose (abbreviated as bTPYglu) and inulin (abbreviated bTPYinu) were used as comparison. Viable cell counts of the bacteria and the pH of the medium were determined during anaerobic incubation period of 0 h, 12 h, 24 h and 48 h at 37 °C. In the presence of carbohydrate source, cultures showed various degree of growth increment. With regards to the growth supporting property: bTPYglu, bTPYglu+glcp, bTPYglcp and bTPYinu were ranked first, second, third and fourth respectively. Interestingly, in bTPYglcp medium, bacterial growth increased with increasing GLCP concentrations when incubated until 24 h. B. longum BB536 was ranked first (10.53 log cfu/mL) in term of their growth in this medium. Growth of B.pseudocatenulatum G4 was ranked second with 10.40 log cfu/mL. This study shows that, GLCP could support the growth of the bacteria tested.

In vitro fermentation of broiler cecal content: the role of lactobacilli and pH value on the composition of microbiota and end products fermentation.

AIM: To assess the probiotic effects of Lactobacillus agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230 and the pH on the cecal microflora of chicken and metabolic end products. METHODS AND RESULTS: An in vitro system, operated with batch bioreactor, was used for this assessment. Selected bacterial species were monitored at two pH values, over 24 h of batch culture incubation. The concentration of short chain fatty acids (SCFA) and lactate in the fermented material was also determined. The addition of L. agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230 into vessel 2 (Cc + P) increased the total anaerobes, lactobacilli and bifidobacteria after 24 h incubation. Moreover, lactobacilli supplementation decreased the total aerobes and streptococci, but it did not have any effects on coliforms. The supplementation of lactobacilli in vessel 2 (Cc + P) was found to significantly increase the production of lactate, propionate and butyrate. Furthermore, pH did not alter the formation of butyrate, whereas the production of acetate and propionate was significantly decreased at pH = 5.8. CONCLUSIONS: L. agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230, as probiotic bacteria, have the ability to re-establish proper microbial balance by the formation of lactate as well as propionate, and stimulate butyrate-producing bacteria to produce butyrate in the chicken cecum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to report this under in vitro conditions, highlighting the probiotic roles of the two Lactobacillus strains in broiler cecal fermentation at different initial pH. These useful data can be helpful in improving the fermentation process in chicken cecum.

Synbiotics growth optimization of Bifidobacterium pseudocatenulatum G4 with prebiotics using a statistical methodology.

AIMS: This study demonstrated the optimum growth of Bifidobacterium pseudocatenulatum G4 with prebiotics via statistical model. METHODS AND RESULTS: Commercial prebiotics [inulin and fructooligosaccharide (FOS)], together with sorbitol, arabinan and inoculum rate, were tested by fractional factorial design to determine their impact on growth of Bif. pseudocatenulatum G4 in skim milk. At 48 h incubation, bacterial growth was mainly influenced by FOS and inoculum rate. Growth reduction was observed in all samples incubated for 72 h. Central composite design (CCD) was adopted using FOS and inoculum rate at 48 h incubation to develop the statistical model for optimization. The model predicted that 2.461 log CFU ml(-1) produced the optimum growth increase of Bif. pseudocatenulatum G4. The combination that produced the optimum point was 2.86% FOS (g/v) and 0.67% inoculum rate (v/v). CONCLUSION: At optimum combination of inoculum rate and FOS, validation experiments recorded 2.40 +/- 10.02 log CFU ml(-1). The application in 1-l bioreactor for 24 h showed higher growth increase of 2.95 log CFU ml(-1). SIGNIFICANT AND IMPACT OF THE STUDY: Response surface methodology approach is useful to develop optimum synbiotics combination for strain G4 with FOS.

Safety evaluation of Bifidobacterium pseudocatenulatum G4 as assessed in BALB/c mice.

AIMS: To assess the safety of Bifidobacterium pseudocatenulatum G4 in BALB/c mice that involves examination of bacterial translocation, changes in the internal organs and histology of the intestinal lining. METHODS AND RESULTS: Forty male BALB/c mice were randomly assigned into five groups (n = 8). Three groups were orally fed with 50 microl of three different concentrations of B. pseudocatenulatum G4 (2 x 10(4), 1 x 10(8) and 1 x 10(11) CFU day(-1)) for 4 weeks. One group was orally administered with 50 microl of 1 x 10(8) CFU B. longum BB536 per day for 4 weeks and last group was used as a nonbifidobacterial treatment control, which received 50 microl of skim milk. The administered strains did not affect the general health of mice and incapable of carrying out translocation to blood or liver. There were no significant differences in the internal organ (liver, heart, kidney and spleen) indices, serum enzymes of liver (aspartate aminotransferase, alkaline phosphate, alanine aminotransferase) and kidney (urea and creatinine) and histology (villi height, crypts height, mucosa thickness and epithelial cell height) of caecum, ileum and colon. CONCLUSION: Administration of high dose of up to 1 x 10(11) CFU B. pseudocatenulatum G4 per day to mice did not show any health threatening symptoms. SIGNIFICANCE AND IMPACT OF THE STUDY: Bifidobacterium pseudocatenulatum G4 is none pathogenic to BALB/c mice and could be safe probiotic for human consumption.

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

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

Antimicrobial susceptibility of bifidobacteria.

Eighteen Bifidobacterium strains were tested for their susceptibility to a range of antimicrobial agents. All the strains tested, including the reference culture Lactobacillus acidophilus CH2, were susceptible to several groups of antimicrobial agents, they were cephalosporin (cefamandole, cefazolin, cefaperazone, cefoxitin), polypeptide (bacitracin), macrolide (erythromycin), penicillin (amoxicillin), phenicol (chloramphenicol) and beta-lactam (imipenem). Fourteen strains were resistant to more than 10 antibiotics. The reference culture was resistant to only three antibiotics. The results showed that bifidobacteria are resistant to a wide range of antimicrobial agents.