Yuan Hong Xie

Effect and Mechanism of Cholesterol-Lowering by Kluyveromyces from Tibetan Kefir

Screening lower high cholesterol and secretion of bile salt hydrolase (BSH) from yeast strains, and study the environmental factors for increasing the activity of BSH, then investigate the mechanism of cholesterol lowering. We obtained two yeasts to lower cholesterol by high-throughput screening technology and o-phthalaldehyde : strain K1 and strain M3 are Kluyveromyces marxianus, are the highest rate of cholesterol-reducing strains by far, the lower cholesterol rates were 68.14% ~ 70.34% , 80.51% ~ 99.12%. The two yeasts can secretion BSH in the metabolic process with the Oxford cup method, and the BSH activity is proportional to the rates of cholesterol-reducing. Strain M3 has a higher BSH production capacity than strain K1, and high cholesterol-lowering effect. That indicating efficient yeast strains to lower cholesterol production is due to their abundant or high activity BSH. The BSH secretion of strain K1 and strain M3 demonstrated a key enzyme in cholesterol-lowering effect for the first time. BSH can hydrolyze conjugated bile salt and free bile salt, which can precipitate with cholesterol state, to reduce the effect of cholesterol. To aim directly at 4 major factors can affect the synthesis of BSH, the fermentation conditions of strain M3 producing BSH highly were determined with four factors and three levels［L9(34)］orthogonal experiment: fermentation temperature is 32 °C, fermentation time is 24h, the initial pH of medium is 6.0, inoculums is 3%. The activity of BSH secreted by Strain M3 increased 50% in the optimization of fermentation conditions. Strain K1 and M3 fermented milk with excellent performance, we could research and develop functional fermented dairy products and micro-ecology preparation, by using the characteristics of efficient to lower cholesterol and complementary advantages of lactose fermentation performance of the two strains.

Purification and Structure Study on Exopolysaccharides Produced by Lactobacillus paracasei KL1-Liu from Tibetan Kefir

To investigate the pure conditions of exopolysaccharides produced by Lactobacillus paracasei KL1-Liu from Tibetan Kefir, and to analyse the structure, we used Multi-level single-factor test to purify EPS by Sepharose CL-6B. And the purity of EPS was detected by UV scan and high performance liquid chromatogram (HPLC). EPS molecular weight and monosaccharide composition were determined by HPLC. Results: Adoption phosphate buffer gradient elution 0.02-0.10 mol/L, the velocity 0.25 mL/min, the sample concentration 1.0 mg/mL, the sample capacity 1.0 mL. Under this purification conditions, components EPSa and EPSb were obtained. The purities of EPSa and EPSb were 82.82% and 91.74% respectively, which were 1.4 and 1.5 times of the pre-purification. Purity Test results showed that EPSa and EPSb polysaccharide were single components, basically no nucleic acid and protein in them. Structural analysis revealed that the molecular weight of EPSa and EPSb were 4.60×104 Da and 2.12×104 Da detected by HPLC. EPSa monosaccharide components were glucose and rhamnose, and the molar ratio was 1:0.68. EPSb were composed of glucose, xylose and rhamnose, and the molar ratio was 1:0.77:0.69.

Optimization of Fermentation Conditions for Higher Monacolin K Production by Monascus purpureus

Monacolin K is a secondary metabolite of Monascus fermentation with lowering blood fat, cholesterol and blood sugar. The culture substrates and fermentation conditions were optimized by means of L9 (33) orthogonal experiments. The optimized fermentation conditions were studied and concluded: initial moisture content 50%, material thickness 4 cm and fermentation time 13 days. Using rice as substrate, the maximum yield of monacolin K was 2.50 mg/g in the optimized conditions. Using glutinousrice as substrate, the maximum yield of monacolin K was 2.71 mg/g in the optimized conditions. The founding determined that glutinousrice was used as substrate in solid state fermentation by Monascus purpureus to produce monacolinK.

Studies on the Resistance of Cholesterol-Lowering of Lactic Acid Bacteria to Simulated Gastrointestinal Circumstance In Vitro

Acid and bile are primary natural barriers in the gastrointestinal tract, and tolerances of the probiotics help to survive and exert their beneficial effects. Eight lactic acid bacteria strains were selected based on cholesterol-lowering ability from Chinese traditional kefir grain, named lactococcus lactis subsp lactis KS4, J4, P1 and P5, lactobacillus casei KTx, KL1 and J1 and streptococcus thermophilus Tx. The strains were cultured in MRS medium (pH 3.0 and 0.3% bile salt). Acid and bile salt tolerances were evaluated by measuring and comparing optical density values at 600nm (OD600). The o-phthalaldehyde method (OPA) was used. The cholesterol-lowering rate of strains KL1 and J1 are 60.1% and 55.3% to simulated gastrointestinal adverse circumstance in vitro. The results indicate that lactobacillus casei KL1 and J1 are effective as probiotics with cholesterol-lowering activities.

The Application of PCR-DGGE Technique in Sheep Intestinal Microbial Diversity Research

To identify the intestinal microflora diversity of transgenic sheep and non-transgenic one with overexpression of foreign antiviral gene (VP1 & SB-SW), DNA of total bacteria were extracted from 16 sheep fecal samples (4 non-transgenic sheep, 6 VP1 transgenic sheep and 6 SB-SW transgenic sheep). And then PCR amplification with bacterial universal primers of V3 variable region of 16S rRNA were taken to get the fingerprint profile by denaturing gradient gel electrophoresis (DGGE) technology. The results showed that the DGGE profiles of the 16 fecal samples were highly polymorphic. The number of DGGE bands, considered indicative of the total species richness, did not vary predictably among the three different samples. The DNA sequences were analyzed and the dominant bacteria in sheep fecal were found to be Bacteroides, Clostridium, and Ruminococcus. Specially, non-transgenic sheep had more Alistipesfinegoldii and Clostridium lentocellum, transgenic sheep with VP1 had more Clostridiumdrakei and Clostridium populeti and transgenic sheep with SB-SW had more Barnesiellaintestinihominis and Clostridium ljungdahlii. So the PCR-DGGE technique can be applied to evaluate genetically modified (GM) animal potential risks to the environment.

Physicochemical Characteristics of Bacteriocin Produced by Pediococcus pentosaceus H6

Lactic acid bacteria H6 was isolated from dried yak jerky by plate dilution method, and using the Listeria monocytogenes as indicator, the Oxford cup method was used to test the bacteriostasis effect. Lactic acid bacteria H6 were identified as Pediococcus pentosaceus by 16S rRNA sequence. The growth curve of Pediococcus pentosaceus H6 was determined by Turbidimetry, and the antibacterial activity of bacteriocin H6 was measured by two fold dilution method and the Oxford cup method. Studying on the thermal stability and pH stability, our results indicated that the barteriocin H6 has better antibacterial effect in acidic conditions or at 100 °C for 90min. The antibacterial spectrum of bacteriocin H6 proved its good inhibitory effect on proliferation of Listeria monocytogenes and other Gram-positive bacteria, and had a certain inhibitory effect on Escherichia coli and other Gram-negative bacteria, showing a broad-spectrum antibacterial property, which demonstrate its great potential in food preservation

Prokaryotic Expression and Purification of Tomato Cnr Protein

Tomato fruit ripening mutant colorless non-ripening (Cnr) is an autosomal dominant mutant, obtained by natural mutation. The fruit of Cnr mutant was obviously immature, and the softening and lycopene accumulation of the Cnr fruit was significantly inhibited during the fruit ripening. In this work, we cloned the Cnr gene from the red ripe tomato fruit by RT-PCR, and constructed a prokaryotic expression vector. And the recombinant Cnr protein was induced by 1 mM IPTG in Escherichia coli BL21 (DE3), and purified by MBP affinity chromatography.

Effect of Bactericine as a Treatment for the Control of Listeria monocytogenes in Refrigerated Meat Products

The aim of this research was to investigate the inhibitory effect of bacteriocin for use as a biological control agent against Listeria monocytogenes in refrigerated meat products. And this bacteriocin was evaluated for their effectiveness as a preservative on pork. The bacteriocin-treated pork was compared with a control pork sample regarding the number of listeria colony count. After 10 days of storage at 4 °C, the population of Listeria monocytogenes was increased from 5.7×105 to 3.7×108 CFU/mL in control samples. In test samples, the population of Listeria monocytogenes underwent a slight increased from 5.7×103 to 6.0×105 CFU/mL. During 10 days of storage, the percentage of growth inhibition ranged from 82.6% to 99.8%, bacteriocin showed their inhibitory effect on Listeria monocytogenes. These results indicated that the studied bacteriocin exhibited bactericidal effect against Listeria monocytogenes strain at refrigerated temperatures, and bacteriocin could be used as antimicrobial agent to preserve the shelf life of refrigerated meat products.

Study on Antifungal Properties of Bacteriocin Produced by Lactobacillus and Application in Fruit Preservation

Strawberry is among the most perishable fruits and vulnerable to physical injuries and fungal invasion. To explore the effects of bacteriocin treatment on strawberry preservation, the inhibitory effect of bacteriocin produced by Lactobacillus on Botrytiscinerea was investigated. Meanwhile, the variation of weight-loss ratio, total soluble solids (TSS) and vitamin C content were determined after treatment of bacteriocin on field-grown strawberry. The results showed that spore germination of B. cinerea was significantly inhibited by bacteriocin at volume percentage of 20%, and bacteriocin inhibited increasing of weight-loss ratio, maintained more ascorbic acid, kept taste of strawberries. Therefore, bacteriocin is a kind of high efficiency natural preservatives and can be applied to the fruit preservation.

Optimization of Liquid Fermentation Conditions and Encapsulation for Nattokinase Production

Nattokinase is a potent fibrinolytic enzyme. The composition of the medium for nattokinase production was optimized respectively. Effect of glucose concentration on nattokinase activity and optimal glucose concentration in fermentation process were investigated. L9 (33) orthogonal array design was applied to investigate the effects of Na2HPO4 to NaH2PO4 ratio, CaCl2, MgSO4 on nattokinase activity and search for the optimal concentrations of the three inorganic salts. The results showed that Na2HPO4/NaH2PO4 was the main factor to affect nattokinase activity, then was CaCl2 and MgSO4 in turn. The optimum concentrations of liquid fermentation medium components were as followed: glucose 1% , Na2HPO4 to NaH2PO4 ratio 0.4% : 0.1% , CaCl2 0.01% and MgSO4 0.1%. Under the optimal conditions, the nattokinase activity was equivalent to 871.56 urokinase IU/mL. To mask the smelly flavor, the nattokinase powders were encapsulated. The research will be helpful to the development of nattokinase products.