Oral administration of Clostridium butyricum for modulating gastrointestinal microflora in mice.

This study aimed to evaluate the safety of Clostridium butyricum and to investigate the effect of C. butyricum on mice ecosystem in the intestinal tract by way of examining the population of different microorganisms isolated from caecal contents. We firstly evaluated the safety of C. butyricum using acute toxicity test and Ames test. Then forty male BALB/c mice were divided into the following four treatment groups, each consisting of ten mice: normal group, low-dose group, medium-dose group and high-dose group. Caecal contents were removed aseptically, immediately placed into an anaerobic chamber, and dissolved in sterile pre-reduced PBS. The determination of Enterococcus spp., Enterobacter spp., Lactobacillus spp., Bifidobacterium spp. and Clostridium perfringens was analyzed by the spread plate method, cell morphologies and biochemical profiles. The results showed the oral maximum tolerated dose of C. butyricum was more than 10 g/kg body weight in mice and no mutagenicity judged by negative experimental results of Ames test. And in medium- and high-dose groups, the populations of Bifidobacterium spp. and Lactobacillus spp. increased in caecum, as well as the ratios of Bifidobacterium spp. and Lactobacillus spp. to Clostridium perfringens (P < 0.01) as compared with the normal group. This research showed the intake of C. butyricum significantly improved the ecosystem of the intestinal tract in BALB/c mice by increasing the amount of probiotics and reducing the populations of unwanted bacteria.

[Construction and confirmation of a recombinant adenovirus vector of survivin].

OBJECTIVE: To construct a recombinant adenovirus of survivin vector and provid valuable reference for gene therapy of laryngeal cancer. METHODS: The survivin gene was cloned by PCR. After confirmation by enzyme restriction analysis and sequencing, the gene and the adenovirus vector were recombined together to construct the recombinant adenovirus vector. The recombinant adenovirus vector was confirmed via both sequencing and digestion restriction analysis, and then linearized and transfected into the HEK 293 cell line to generate recombinant adenovirus. RESULTS: The sequence analysis demonstrated that the survivin gene sequence was the same as published in the literature, suggesting that a recombinant adenovirus vector has been successfully constructed. CONCLUSIONS: A survivin recombinant adenovirus has been successfully constructed.

Effect of ultrasonic-ionic liquid pretreatment on the hydrolysis degree and antigenicity of enzymatic hydrolysates from whey protein.

With the aim to reduce the antigenicity of whey protein hydrolysate in milk, the pretreatment method of coupling ultrasonic and ionic liquid (US-IL) and further enzymatic treatments were studied. Papain and alcalase were found to be suitable for ultrasonic-ionic liquid pretreatment. After ultrasound-ionic liquid treatment, the antigenic decline rates of ALA and BLG upon alcalase hydrolysis were 82.82% and 88.01%, and that of the papain hydrolysis was 81.87% and 88.46%, respectively. Upon ultrasonic-ionic liquid pretreatment, the molecular weight of whey protein did not change significantly, but the small molecular weight proportion of components in the enzymatic hydrolysate obviously increased. The findings showed that combining with US-IL pretreatment for further protease hydrolysis of whey proteins, the hydrolysate can be used in order to produce hypoallergenic bovine whey proteins.

[Evaluation of compliance with bundle treatment in the management of severe infection].

OBJECTIVE: To evaluate compliance with bundle treatment in the management of severe infection in a tertiary hospital, aiming at analyzing clinical data in order to popularize guidelines for management of severe sepsis and septic shock. METHODS: A 14-month (from November 1, 2006 to December 31, 2007) prospective observational study of a group of 43 patients admitted to the respiratory intensive care unit in First Affiliated Hospital (tertiary hospital) of Guangzhou Medical College meeting the criteria for severe pneumonia and septic shock was carried out. Implementation of 6-hour and 24-hour bundle treatment for severe infection was divided into three phases consisting of education, trial, and application. A cohort of 43 patients with matched disease history admitted during January 1, 2004 to October 31, 2006 were enrolled as control group. RESULTS: (1) In 6-hour bundle treatment for severe infection, 20.9% (9/43) had serum lactate measured, blood culture was obtained prior to antibiotic administration in 7.0% (3/43) of patients, 100% (43/43) had empirical antibiotics administration within 1 hour, an infusion of an initial minimum of 20 ml/kg of crystalloid or colloid equivalent (1.1 ml/kg of 20% albumin or 4.8 ml/kg of 6% hydroxyethyl starch) was given in 44.2% (19/43), with infused fluid (converted into 6% hydroxyethyl starch) reaching (503.95+/-176.19) ml within 6 hours, in 94.7% (18/19) of patients had received vasopressors , and inotropic dobutamine and/or transfusion of packed red blood cells were administered in 7.0% (3/43). (2) In 24-hour bundle treatment for severe infection group, 31.6% (6/19) had received low-dose steroids, 34.9% (15/43) had their blood glucose controlled<8.3 mmol/L, mechanical ventilation with inspiratory plateau pressures maintained<30 cm H(2)O (1 cm H(2)O=0.098 kPa, 6 ml/kg tidal volume) was instituted in 97.6% (40/41) of patients. (3) The percentage of compliance with 6-hour and 24-hour bundle treatment for severe infection were 0 and 21.4% respectively, total compliance was also 0. (4) As compared with control group, a 23.30% absolute mortality reduction was found in bundle group (18.6% vs. 41.9%, P=0.019). CONCLUSION: Bundle treatment for severe infection is complied with partially in our hospital, suggesting that it is still quite arduous to popularize guidelines for management of severe sepsis and septic shock in our country.

Investigations of the cytotoxicity of epigallocatechin-3-gallate against PC-3 cells in the presence of Cd2+ in vitro.

The epidemiological studies and recent data have provided convinced evidence that green tea and its major constituent epigallocatechin gallate (EGCG) might have the potential to lower the risk of cancers in humans. Metal ions, such as zinc and cadmium, which are necessary to our health, are important factors inducing many diseases including prostate cancer in the condition of absence or excess. EGCG can satisfactorily exhibit complex chemistry with metal ions because of multiple hydroxyl states, which in turn changes their bioactivities and metabolism pathways. This paper presents the results of an investigation of the cytotoxicity of EGCG against PC-3 prostate cancer cells in the presence and absence of Cd2+ in vitro. The results showed that both EGCG and Cd2+ suppressed viability and clonegenecity of PC-3 cells, and the suppression effect was enhanced when EGCG added with Cd2+. Although Cd2+ up-regulated the 67 kDa laminin receptor (67LR), which is a migration-associated protein, the cell migration ability was not significantly increased after each treatment. We also found that EGCG and Cd2+ directly interacted with mitochondrial, and the mixture of EGCG and Cd2+ (EGCG+Cd2+) significantly caused loss of the mitochondrial membrane potential, decrease of the ATP content and activation of caspase-9 compared with EGCG treated alone. Taken together, these findings suggest that Cd2+ enhanced the cytotoxicity of EGCG to PC-3 cells by up-regulating the 67LR and the mitochondria-mediated apoptosis pathway.

Occurrence of bound deoxynivalenol in Fusarium head blight-infected barley (Hordeum vulgare L.) and malt as determined by solvolysis with trifluoroacetic acid.

Fusarium head blight occurs worldwide in barley production, and the Fusarium mycotoxins, particularly deoxynivalenol (DON), have become a major concern for barley products, such as beer. This study investigated the content of bound DON in barley samples that were naturally infected with Fusarium head blight. Free DON was determined by gas chromatography-electron capture detection after standard acetonitrile:water extraction, while total DON was determined using treatment with trifluoroacetic acid. Bound DON is the difference between the total and free values. Bound DON was detected in approximately 40% of the samples analyzed, and represented 6 to 21% of free DON. A preliminary study indicated that both free and bound DON decrease significantly during the steeping phase of malting. These results suggest that bound DON, like the free form, may be water-soluble.

Optimization of acidic extraction of astaxanthin from Phaffia rhodozyma.

Optimization of a process for extracting astaxanthin from Phaffia rhodozyma by acidic method was investigated, regarding several extraction factors such as acids, organic solvents, temperature and time. Fractional factorial design, central composite design and response surface methodology were used to derive a statistically optimal model, which corresponded to the following optimal condition: concentration of lactic acid at 5.55 mol/L, ratio of ethanol to yeast dry weight at 20.25 ml/g, temperature for cell-disruption at 30 degrees C, and extraction time for 3 min. Under this condition, astaxanthin and the total carotenoids could be extracted in amounts of 1294.7 microg/g and 1516.0 microg/g, respectively. This acidic method has advantages such as high extraction efficiency, low chemical toxicity and no special requirement of instruments. Therefore, it might be a more feasible and practical method for industrial practice.

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination.

The bglS gene encoding endo-l,3-1,4-beta-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone alpha-factor (MFalpha1(S)), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-betaG) was preliminarily screened by the clearing hydrolysis zone formed after the barley beta-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-beta-glucanase assay methods showed that the recombinant strain SC-betaG had high endo-l,3-1,4-beta-glucanase expression level with the maximum of 69.3 U/(h.ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-beta-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Spoilage potential of psychrotrophic bacteria isolated from raw milk and the thermo-stability of their enzymes.

The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive ß-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 °C, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.

Comparison of effects of epigallocatechin-3-gallate on hypoxia injury to human umbilical vein, RF/6A, and ECV304 cells induced by Na(2)S(2)O(4).

Hypoxia is related to the etiology of numerous pathological disease states, such as the formation of tumors or diverse retinopathies. Epigallocatechin-3-gallate (EGCG), a potent polyphenolic antioxidant and antiangiogenic compound found in green tea, has been shown to suppress the growth of blood vessels necessary for the growth of tumors and the induction of retinopathies. However, only a few studies have been carried focusing on the protective effects of EGCG on hypoxia-induced injury of cultured endothelial cells. The present study investigated the effects of EGCG on Na(2)S(2)O(4)-induced hypoxic injury in three types of cultured endothelial cells, primary isolates of normal human umbilical vein endothelial cells (HUVECs), and two transformed endothelial cells lines, RF/6A and ECV304. Our results indicated that Na(2)S(2)O(4) inhibited the growth of HUVE, RF/6A, and ECV304 cells in a dose-dependent manner; EGCG also exerted inhibitory effects on the growth of the three cell types, but the toxicity of EGCG to HUVECs was less than to RF/6A and ECV304 cells. The viability of HUVE, RF/6A, and ECV304 cells treated with EGGC were the lowest at 24, 24, and 36 h, respectively, and the IC(50) of EGCG were 420 +/- 8.0, 125 +/- 7.1, and 75 +/- 5.1 microM, respectively. Furthermore, EGCG, an efficient nontoxic agent, protected all three cell types from Na(2)S(2)O(4)-induced hypoxia injury, providing partial protection from hypoxia-induced injury in normal endothelial cells at 100, 30, and 10 microM for HUVE, RF/6A, and ECV304 cells, respectively.

Doehlert matrix design for optimization of the determination of bound deoxynivalenol in barley grain with trifluoroacetic acid (TFA).

Fusarium head blight (FHB) is an impediment to barley production in many regions of the world. Tricothecene toxins, associated with FHB-infected grain, particularly, deoxynivalenol (DON), pose a serious threat to human and animal health. Recent research has suggested that a portion of the DON present on grain is bound and escapes detection through conventional determination. The objective of this study was to optimize a method for determination of nonextractable DON in barley grain using trifluoroacetic acid (TFA). A Doehlert matrix design was performed to determine the optimal conditions for time, temperature, and TFA concentration. These conditions were treated with 1.25 N TFA in 86:14 acetontrile/water for 54 min at 133 degrees C. Cleanup, derivatization, and determination of DON by a gas chromatography electron capture detector (GC-ECD) was as normal. Treatment of the test sample resulted in the release of an additional 58% DON under the optimized conditions and an increase of 9-88% in a set of verification samples.

Effect of cultivating conditions on alpha-galactosidase production by a novel Aspergillus foetidus ZU-G1 strain in solid-state fermentation.

The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Aspergillus foetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of alpha-galactosidase production in SSF were 60% initial moisture of medium, 28 degrees C incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0 approximately 6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum alpha-galactosidase production was 2 037.51 U/g dry matter near the 144th hour of fermentation.

Enhanced production of elastase by Bacillus licheniformis ZJUEL31410: optimization of cultivation conditions using response surface methodology.

Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cultures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.

Studies on optimization of nitrogen sources for astaxanthin production by Phaffia rhodozyma.

Fermentation of Phaffia rhodozyma is a major method for producing astaxanthin, an important pigment with industrial and pharmaceutical application. To improve astaxanthin productivity, single factor and mixture design experiments were used to investigate the effects of nitrogen source on Phaffia rhodozyma cultivation and astaxanthin production. Results of single factor experiments showed nitrogen source could significantly affect P. rhodozyma cultivation with respect to carbon source utilization, yeast growth and astaxanthin accumulation. Further studies of mixture design experiments using (NH(4))(2)SO(4), KNO(3) and beef extract as nitrogen sources indicated that the proportion of three nitrogen sources was very important to astaxanthin production. Validation experiments showed that the optimal nitrogen source was composed of 0.28 g/L (NH(4))(2)SO(4), 0.49 g/L KNO(3) and 1.19 g/L beef extract. The kinetic characteristics of batch cultivation were investigated in a 5-L pH-stat fermentor. The maximum amount of biomass and highest astaxanthin yield in terms of volume and in terms of biomass were 7.71 mg/L and 1.00 mg/g, respectively.

Apoptosis induction of colorectal cancer cells HTL-9 in vitro by the transformed products of soybean isoflavones by Ganoderma lucidum.

Soybean isoflavones have been one of the potential preventive candidates for antitumor research in recent years. In this paper, we first studied the transformation of soybean isoflavones with the homogenized slurry of Ganoderma lucidum. The resultant transformed products (TSI) contained (703.21±4.35) mg/g of genistein, with transformed rates of 96.63% and 87.82% of daidzein and genistein, respectively, and TSI also could enrich the bioactive metabolites of G. lucidum. The antitumor effects of TSI on human colorectal cancer cell line HTL-9, human breast cancer cell line MCF-7, and human immortalized gastric epithelial cell line GES-1 were also studied. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay showed that TSI could dramatically reduce the viability rates of HTL-9 cells and MCF-7 cells without detectable cytotoxicity on GES-1 normal cells when the TSI concentration was lower than 100 µg/ml. With 100 µg/ml of TSI, HTL-9 cells were arrested in the G1 phase, and late-apoptosis was primarily induced, accompanied with partial early-apoptosis. TSI could induce primarily early-apoptosis by arresting cells in the G1 phase of MCF-7 cells. For HTL-9 cells, Western-blot and reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that TSI (100 µg/ml) can up-regulate the expression of Bax, Caspase-3, Caspase-8, and cytochrome c (Cyto-c), indicating that TSI could induce cell apoptosis mainly through the mitochondrial pathway. In addition, the expression of p53 was up-regulated, while the expression of Survivin and nuclear factor κB (NF-κB) was down-regulated. All these results showed that TSI could induce apoptosis of HTL-9 cells by the regulation of multiple apoptosis-related genes.

Phenotypic and genetic heterogeneity within biofilms with particular emphasis on persistence and antimicrobial tolerance.

Phenotypic changes or phase variation within biofilms is an important feature of bacterial dormant life. Enhanced resistance to antimicrobials is one of the distinct features displayed by a fraction of cells within biofilms. It is believed that persisters are mainly responsible for this phenotypic heterogeneity. However, there is still an unresolved debate on the formation of persisters. In this short review, we highlight all known genomic and proteomic changes encountered by bacterial cells within biofilms. We have also described all phenotypic changes displayed by bacterial cells within biofilms with particular emphasis on enhanced antimicrobial tolerance of biofilms with particular reference to persisters. In addition, all currently known models of persistence have been succinctly discussed.

Insights into the microbial diversity and community dynamics of Chinese traditional fermented foods from using high-throughput sequencing approaches.

Chinese traditional fermented foods have a very long history dating back thousands of years and have become an indispensable part of Chinese dietary culture. A plethora of research has been conducted to unravel the composition and dynamics of microbial consortia associated with Chinese traditional fermented foods using culture-dependent as well as culture-independent methods, like different high-throughput sequencing (HTS) techniques. These HTS techniques enable us to understand the relationship between a food product and its microbes to a greater extent than ever before. Considering the importance of Chinese traditional fermented products, the objective of this paper is to review the diversity and dynamics of microbiota in Chinese traditional fermented foods revealed by HTS approaches.

Octenyl succinic anhydride modified early indica rice starches differing in amylose content.

Seven early indica rice starches with different amylose contents were modified by octenyl succinic anhydride (OSA) in aqueous suspension systems to evaluate the effect of amylose contents on starch esterification. The crystalline structure and pasting properties of starches were investigated using X-ray diffraction and a Rapid Visco Analyzer (RVA). The results indicated that the amylose content had a positive impact on the OSA modification. As the amylose content increased from 0 to 39.6%, the degree of substitution increased from 0.024 to 0.030 and the reaction efficiency increased from 62.8 to 77.5%. X-ray diffraction scans confirmed that the amylose was mainly present in the amorphous domain of the granule and was highly substituted after the OSA treatment. The RVA profiles demonstrated that the OSA starches had higher viscosities than their native counterparts. Moreover, negative correlations were observed between the amylose content and the major RVA parameters (e.g., peak viscosity, hot paste viscosity, cool paste viscosity, and breakdown viscosity).

Optimization of elastolysis conditions and elastolytic kinetic analysis with elastase from Bacillus licheniformis ZJUEL31410.

The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows: elastolysis temperature 50 degrees C, elastase concentration 1 x 10(4) U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasing elastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods.

Synthesis and paste properties of octenyl succinic anhydride modified early Indica rice starch.

Octenyl succinic anhydride (OSA) modified early Indica rice starch was prepared in aqueous slurry systems using response surface methodology. The paste properties of the OSA starch were also investigated. Results indicated that the suitable parameters for the preparation of OSA starch from early Indica rice starch were as follows: reaction period 4 h, reaction temperature 33.4 degrees C, pH of reaction system 8.4, concentration of starch slurry 36.8% (in proportion to water, w/w), amount of OSA 3% (in proportion to starch, w/w). The degree of substitution was 0.0188 and the reaction efficiency was 81.0%. The results of paste properties showed that with increased OSA modification, the starch derivatives had higher paste clarity, decreased retrogradation and better freeze-thaw stability.