The polysaccharides from seeds of Glycyrrhiza uralensis ameliorate metabolic disorders and restructure gut microbiota in type 2 diabetic mice.

T2D and its complications are significant threats to human health and are among the most concerning metabolic diseases worldwide. Previous studies have revealed that Glycyrrhiza uralensis polysaccharide extract (GUP) exhibits remarkable antioxidant capabilities and inhibits alpha-glucosidase activity. However, whether GUP improves glycemic control in T2D is unknown. This study aims to investigate the effects of GUP on glucose and lipid metabolism as well as the intestinal microbiota in HFD/STZ-induced T2D. The results demonstrated that GUP could significantly ameliorate hyperglycemia, insulin resistance, oxidative stress, and reduce liver lipid levels in T2D mice. Furthermore, it also enhanced the integrity of the intestinal barrier in T2D mice by reducing the levels of pro-inflammatory cytokines and serum LPS levels. Interestingly, GUP treatment significantly lowered serum creatinine and urea nitrogen levels, mitigating renal function deterioration and interstitial fibrosis. Additionally, GUP intervention increased the α diversity of gut microbiota, promoting beneficial species like Akkermansia, Lactobacillus, Romboutsia and Faecalibaculum, while decreasing harmful ones such as Bacteroides, Escherichia-Shigella, and Clostridium sensu stricto 1 in T2D mice. Overall, this study highlights the potential of GUP in alleviating complications and enhancing intestinal health in T2D mice, providing valuable insights into dietary strategies for diabetes control and overall health improvement.

Succinylated starch emulsified Eugenol and Carvacrol nanoemulsions improved digestive stability, bio-accessibility and Salmonella typhimurium inhibition.

The ultrasonically processed Eugenol (EU) and Carvacrol (CAR) nanoemulsions (NE) were successfully optimized via response surface methodology (RSM) to achieve broad spectrum antimicrobial efficacy. These NE were prepared using 2 % (w/w) purity gum ultra (i.e., succinylated starch), 10 % (v/v) oil phase, 80 % (800 W) sonication power, and 10 min of processing time as determined via RSM. The second order Polynomial method was suitable to RSM with a co-efficient of determination >0.90 and a narrow polydispersity index (PDI) ranging 0.12-0.19. NE had small droplet sizes (135.5-160 nm) and low volatility at high temperatures. The EU & CAR entrapment and heat stability (300 °C) confirmed by Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). Further, the volatility of EU & CAR NE was 18.18 ± 0.13 % and 12.29 ± 0.11 % respectively, being lower than that of bulk/unencapsulated EU & CAR (i.e., 23.48 ± 0.38 % and 19.11 ± 0.08 %) after 2 h at 90 °C. Interestingly, both EU & CAR NE showed sustained release behaviour till 48 h. Their digest could inhibit Salmonella typhimurium (S. typhimurium) via membrane disruption and access to cellular machinery as evident from SEM images. Furthermore, in-vivo bio-accessibility of EU & CAR in mice serum was up to 80 %. These cost-effective and short-processed EU/CAR NE have the potential as green preservatives for food industry.

Size-Related Pathway Flux Analysis of Ultrasmall Iron Oxide Nanoparticles in Macrophage Cell RAW264.7 for Safety Evaluation.

The endocytosis, intracellular transport, and exocytosis of different-sized nanoparticles were reported to greatly affect their efficacy and biosafety. The quantitation of endocytosis and exocytosis as well as subcellular distribution of nanoparticles might be an effective approach based on transport pathway flux analysis. Thus, the key parameters that could present the effects of three different-sized ultrasmall iron oxide nanoparticles (USIONPs) were systematically investigated in RAW264.7 cells. The endocytosis and exocytosis of USIONPs were related to their sizes; 15.4 nm of S2 could be quickly and more internalized and excreted in comparison to S1 (7.8 nm) and S3 (30.7 nm). In RAW264.7 cells, USIONPs were observed in endosomes, lysosomes, the Golgi apparatus, and autophagosomes via a transmission electron microscope. Based on flux analysis of intracellular transport pathways of USIONPs, it was found that 43% of S1, 40% of S2, and 44% of S3 were individually transported extracellularly through the Golgi apparatus-involved middle-fast pathway, while 24% of S1, 23% of S2, and 26% of S3 were transported through the fast recycling endosomal pathway, and the residues were transported through the slower speed lysosomal pathway. USIONPs might be transported via size-related endocytosis and exocytosis pathways. The pathway flux could be calculated on the basis of disturbance analysis of special transporters as well as their coding genes. Because there were rate differences among these transport pathways, this pathway flux could anticipate the intracellular remaining time and distribution of different-sized nanoparticles, the function exertion, and side effects of nanomaterials. The size of the nanomaterials could be optimized for improving functions and safety.

Phage cocktail alleviated type 2 diabetes by reshaping gut microbiota and decreasing proinflammatory cytokines.

Type 2 diabetes (T2D), a global health concern, is closely associated with the gut microbiota. Restoration of a balanced microbiota and intestinal homeostasis benefit therapy of T2D. Some special phages may selectively alter the gut microbiota without causing dysbiosis, such as MS2 and P22. However, scarcely systematic analysis of cascading effects triggered by MS2 and P22 phages on the microbiota, as well as interactions between specific gut bacteria and systemic metabolism, seriously inhibit the development of positive interventions of phages. Based on multi-omic analysis, we analyzed the intrinsic correlations among specific microbiota, their bioactive metabolites, and key indicators of T2D. We found that gavage of the MS2-P22 phage cocktail could significantly alter the gut microbiome to attenuate dysbiosis of diabetic C57BL/6 mice caused by high-fat diets (HFDs) and streptozotocin (STZ), by affecting microbial compositions as well as their metabolic pathways and metabolites, especially increasing amounts of short-chain fatty acid-producing (SCFA-producing) bacteria (e.g., Blautia and Romboutsia) and short-chain fatty acids (SCFAs). Correspondingly, a noteworthy reduction in the number of several opportunistic pathogens occurred, e.g., Candidatus Saccharimonas, Aerococcus, Oscillibacter, Desulfovibrio, and Clostridium sensu stricto 1. Synchronously, the levels of proinflammatory cytokines and lipopolysaccharide (LPS) were reduced to recover gut barrier function in T2D mice. These findings might benefit the development of a new dietary intervention for T2D based on phage cocktails. KEY POINTS: • Intestinal barrier integrity of T2D mice is improved by a phage cocktail • Negative relationship between Muribaculaceae and Corynebacterium reshaped gut microbiota • Acetate, propionate, and butyrate decreased the level of proinflammatory factors.

Positive interactions among Corynebacterium glutamicum and keystone bacteria producing SCFAs benefited T2D mice to rebuild gut eubiosis.

Accumulating evidences strongly support the correlations between the compositions of gut microbiome and therapeutic effects on Type 2 diabetes (T2D). Notably, gut microbes such as Akkermansia muciniphila are found able to regulate microecological balance and alleviate dysmetabolism of mice bearing T2D. In order to search out similarly functional bacteria, bacteriophage MS2 with a good specificity to bacteria carrying fertility (F) factor were used to treat T2D mice. Based on multi-omics analysis of microbiome and global metabolism of mice, we observed that gavage of bacteriophage MS2 and metformin led to a significant increase in the abundance of Corynebacterium glutamicum and A. muciniphila, respectively. Consequently, the gut microbiota were remodeled, leading to variations in metabolites and a substantial increase in short-chain fatty acids (SCFAs). In which, the amount of acetate, propionate, and butyrate presented negative correlations to that of proinflammatory cytokines, which was beneficial to repairing the intestinal barriers and improving their functions. Moreover, main short fatty acid (SCFA) producers exhibited positive interactions, further facilitating the restoration of gut eubiosis. These findings revealed that C. glutamicum and its metabolites may be potential dietary supplements for the treatment of T2D. Moreover, our research contributes to a novel understanding of the underlying mechanism by which functional foods exert their anti-diabetic effects.

DNA binding peptide CF-14 enhances bactericidal efficacy of eugenol/carvacrol nanoparticles to Escherichia coli.

To evaluate the bactericidal action of antimicrobial peptide CF-14, Eugenol (EU) and carvacrol (CAR) nanoparticles (NPs) less than 200 nm were surface-modified with CF14, gaining approximately 200 nm of EU-CF and CAR-CF NPs with swollen morphology. EU-CF and CAR-CF NPs were bactericidal to E. coli at dosage of 0.09% and 0.07% (v/v), respectively; while they were just bacteriostatic to Staphylococcus aureus at 0.10% and 0.08% (v/v). Spectral variations in bacterial carbohydrates (1185-900 cm-1), lipids (3000-2800 cm-1) and DNA (1500-1185 cm-1) were obvious as evident from Fourier transform infrared spectroscopy (FTIR). A higher percentage of membrane damaged (non-revivable) E. coli than S. aureus was found, which indicated electrostatic interactions between Gram-negative E. coli with cationic CF conjugated NPs leading to DNA disintegration. Interestingly, EU-CF and CAR-CF NPs inhibited E. coli growth in orange juice without impacting flavour compounds.

Obesity, but not high-fat diet, is associated with bone loss that is reversed via CD4+CD25+Foxp3+ Tregs-mediated gut microbiome of non-obese mice.

Osteoporosis is characterized by decreased bone mass, microarchitectural deterioration, and increased bone fragility. High-fat diet (HFD)-induced obesity also results in bone loss, which is associated with an imbalanced gut microbiome. However, whether HFD-induced obesity or HFD itself promotes osteoclastogenesis and consequent bone loss remains unclear. In this study, we developed HFD-induced obesity (HIO) and non-obesity (NO) mouse models to evaluate the effect of HFD on bone loss. NO mice were defined as body weight within 5% of higher or lower than that of chow diet fed mice after 10 weeks HFD feeding. NO was protected from HIO-induced bone loss by the RANKL /OPG system, with associated increases in the tibia tenacity, cortical bone mean density, bone volume of cancellous bone, and trabecular number. This led to increased bone strength and improved bone microstructure via the microbiome-short-chain fatty acids (SCFAs) regulation. Additionally, endogenous gut-SCFAs produced by the NO mice activated free fatty acid receptor 2 and inhibited histone deacetylases, resulting in the promotion of Treg cell proliferation in the HFD-fed NO mice; thereby, inhibiting osteoclastogenesis, which can be transplanted by fecal microbiome. Furthermore, T cells from NO mice retain differentiation of osteoclast precursors of RAW 264.7 macrophages ex vivo. Our data reveal that HFD is not a deleterious diet; however, the induction of obesity serves as a key trigger of bone loss that can be blocked by a NO mouse-specific gut microbiome.

Label-free fluorescence aptasensor for the detection of patulin using target-induced DNA gates and TCPP/BDC-NH2 mixed ligands functionalized Zr-MOF systems.

Patulin (PAT) is an unsaturated lactone mycotoxin primarily produced by Penicillium expansum and Aspergillus clavatus. Given the potential health risks and economic losses associated with PAT, the rapid detection of PAT using fluorescent aptasensors is of significant importance in evaluating food safety. However, it easily increases the cost and complexity caused by signal labeling. We combined TCPP/BDC-NH2 mixed ligands functionalized Zr metal-organic frameworks (Zr-MOFmix) and terminated three-stranded DNA gates (ttsDNA gates) to fabricate a label-free fluorescent aptasensor for PAT detection. The Zr-MOFmix system was synthesized via a one-pot strategy and could be used to address the problem of pore size limitation and increase the loading amounts of dyes. TtsDNA gate was integrated into the Zr-MOFmix system to control the release of dyes, exhibiting a high signal-to-background ratio. The single-stranded aptamer region in ttsDNA gate situated away from the surface of the Zr-MOFmix, resulting in a natural release of dyes in the absence of PAT. While binding to PAT resulted in target-induced conformational changes that helped form the hairpin structure of the aptamer. This structure hindered the release of dyes from the pores of Zr-MOFmix, thus reducing the fluorescence signals intensity. The stimuli-responsive DNA-gated material provides a platform for PAT analysis under conditions of a low limit of detection (0.871 pg/mL). Furthermore, the excellent specificity and anti-interference of the fluorescent aptasensor make the system suitable for the analysis of apple juice samples. This label-free strategy is cheaper and simper compared with labeled detection, especially for the development of multi-target-detection.

Phage-based technologies for highly sensitive luminescent detection of foodborne pathogens and microbial toxins: A review.

Foodborne pathogens and microbial toxins are the main causes of foodborne illness. However, trace pathogens and toxins in foods are difficult to detect. Thus, techniques for their rapid and sensitive identification and quantification are urgently needed. Phages can specifically recognize and adhere to certain species of microbes or toxins due to molecular complementation between capsid proteins of phages and receptors on the host cell wall or toxins, and thus they have been successfully developed into a detection platform for pathogens and toxins. This review presents an update on phage-based luminescent detection technologies as well as their working principles and characteristics. Based on phage display techniques of temperate phages, reporter gene detection assays have been designed to sensitively detect trace pathogens by luminous intensity. By the host-specific lytic effects of virulent phages, enzyme-catalyzed chemiluminescent detection technologies for pathogens have been exploited. Notably, these phage-based luminescent detection technologies can discriminate viable versus dead microbes. Further, highly selective and sensitive immune-based assays have been developed to detect trace toxins qualitatively and quantitatively via antibody analogs displayed by phages, such as phage-ELISA (enzyme-linked immunosorbent assay) and phage-IPCR (immuno-polymerase chain reaction). This literature research may lead to novel and innocuous phage-based rapid detection technologies to ensure food safety.

Metagenomic analysis of microflora structure and functional capacity in probiotic Tibetan kefir grains.

Tibetan kefir grains (TKGs) are distinctive and complex mixtures with protein-lipid-polysaccharide matrices and multiple microorganism species. The objective of this study was to evaluate the microflora composition, probiotic species and functional genes within TKGs. Metagenomic analysis was used to evaluate communities of three TKGs, revealing the presence of 715 species, with Lactobacillus kefiranofaciens as the most dominant species. The relative abundances of acetic acid bacteria and yeast significantly differed among the three TKGs (acetic acid bacteria: p < 0.01; yeast: p < 0.05), and the dominant yeast species also varied across three TKGs. Lactobacillus helveticus was the most abundant listed probiotic species, and its abundance did not significantly differ across three TKGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that ko01501 was the most abundant pathway that related to human disease. There are 16 different KOs (KEGG Orthology) in the ko01501 pathway were annotated in TKGs, which helps to resist ß-lactam. This study provided a new insight into the microbial community structures and the presence of probiotic species within TKGs and provides a foundation for further targeted studies.

Design and Preparation of "corn-like" SPIONs@DFK-SBP-M13 Assembly for Improvement of Effective Internalization.

PURPOSE: Superparamagnetic iron oxide nanoparticles (SPIONs) have exhibited preeminent diagnosis and treatment performances, but their low internalization severely limits predesigned functions. The low cell internalization is now an urgent bottleneck problem for almost all nanomaterials. To achieve more internalization of SPIONS, recombinant M13 phage was designed for targeted delivery and smart release. METHODS: M13 phages were designed to co-express exogenous SPARC binding peptide (SBP) and cathepsin B cleavage peptide (DFK), formed recombinant DFK-SBP-M13. 3.37± 0.06 nm of SPIONs were modified by 3, 4-dihydroxyhydrocinnamic acid (DHCA) to gain 10.80 ± 0.21 nm of DHCA-coated SPIONs, i.e., DHCA@SPIONs. Upon adjusting the proportions of DHCA@SPIONs and DFK-SBP-M13, the multi-carboxyl SPIONs assembled onto recombinant M13 phages via covalent bonding. The assemblies were co-cultured with MDA-MB-231 cells to interpret their internalization and smart release. RESULTS: The "corn-like" SPIONs@DFK-SBP-M13 (261.47±3.30 nm) assemblies have not been reported previously. The assembly was stable, dispersible, superparamagnetic and biocompatible. After co-cultivation with MDA-MB-231 cells, the SPIONs@DFK-SBP-M13 assemblies quickly bond to the cell surface and are internalized. The enrichment rate of SPIONs@DFK-SBP-M13 assembly was 13.9 times higher than free SPIONs at 0.5 h, and intracellular Fe content was 3.6 times higher at 1 h. Furthermore, the DFK peptides favored cathepsin B to cleave SPIONs from the M13 templates resulting in release of SPIONs inside cells. CONCLUSION: The novel SPIONs@DFK-SBP-M13 assembly can rapidly deliver SPIONs to the targeted sites and enabled smart release. The combination of genetic recombination and nanotechnology is beneficial for designing and optimizing some new nanomaterials with special functions to achieve wider applications.

The toxicity mechanism of different sized iron nanoparticles on human breast cancer (MCF7) cells.

The toxicity mechanism of superparamagnetic iron oxide nanoparticles (SPIONs) were examined multidimensionally to reduce the toxicity risks. A higher dosage and more suitable size of SPIONs enhanced the uptake amount into MCF7 cells, leading to a higher specific uptake rate of SPIONs with the formation of more reactive oxygen species (ROS). ROS was an intrinsic factor of cell death. Interestingly, the smaller SPIONs (S1) liked to produce more ROS in mitochondria to damage mitochondria, while the larger SPIONs (S2 and S3) promoted ROS yield in plasma to destroy cytomembrane. Furthermore, ROS synthesis pathways were the partial of cell death pathways, and ferroptosis pathway was the main contributor to mitochondrial and cytomembrane damage. Meanwhile, ROS amount was well coincided to gene expression level of these cell death pathways, which inferred RNA-seq might be a new method to evaluate the oxidative stress and potential toxicity of nanomaterials.

Fluorescent recognition and selective detection of nitrite ions with carbon quantum dots.

The nitrite ion (NO2-) is a vital inorganic species that occurs both in natural ecological systems and human bodies. The high concentration of NO2- can be harmful for animal and human health. It is important to develop a simple, sensitive, reliable, and economic methodology to precisely monitor NO2- in various environmental and biological fields. Thus, a novel nitrite biosensor based on carbon quantum dots (PA-CDs) has been constructed and prepared via a high-efficiency, one-pot hydrothermal route using primary arylamines (PA) such as m-phenylenediamine. The device exhibits bright green fluorescence and a high quantum yield of 20.1% in water. In addition, the PA-CDs also possess two broad linear ranges: 0.05-1.0 µM and 1.0-50 µM with a low detection limit of 7.1 nM. The classical diazo reaction is firstly integrated into the PA-CD system by primary arylamines, which endows the system with high sensitivity and specific selectivity towards nitrite. Importantly, the nanosensor can detect NO2- in environmental water and serum samples with high fluorescence recoveries, demonstrating its feasibility in practical applications. This work broadens a new method to fabricate novel nanosensors and provides a prospective application for fluorescent carbon quantum dots (CDs). Graphical abstract.

Mechanism and intervention measures of iron side effects on the intestine.

Excess oral iron in the intestinal tract usually produces reactive oxygen species via Fenton and Haber-Weiss reaction, so oxidative stress is triggered. Lipid peroxidation procedurally appears, ferroptosis, apoptosis and necrosis are often induced, subsequently, mitochondrial damage, endoplasmic reticulum dysfunction and even cell death occur. As a result, the intestinal epithelial cells are destroyed, leading to the incompleteness of intestinal mechanical barrier. Simultaneously, iron supplement can change the compositions and metabolic processes of intestinal microbes, and the intestinal inflammatory may be worsened. In principle, the easier dissociation of Fe2+ from oral iron supplements is, the more serious intestinal inflammation will occur. Fortunately, some interventions have been developed to alleviate these side effects. For instance, some antioxidants e.g. VE and ferulic acid have been used to prevent the formation of free radicals or to neutralize the formed free radicals. Furthermore, some new iron supplements with the ability of slow-releasing Fe2+, e.g. ferrous citrate liposome and EDTA iron sodium, have been successfully prepared. In order to recover the intestinal micro-ecological balance, probiotics and prebiotics, bacterial consortium transplantation, and fecal microbiota transplantation have been developed. This study is meaningful for us to develop safer oral iron supplements and to maintain intestinal micro-ecological health.

A Probe for Fluorescence Detection of the Acetylcholinesterase Activity Based on Molecularly Imprinted Polymers Coated Carbon Dots.

This paper presents a new probe for fluorescence detection of the acetylcholinesterase (AChE) activity based on molecularly imprinted polymer (MIP) coated carbon dots (C-dots) composite. The C-dots were hydrothermally synthesized with grafted silica surface and sealed with molecularly imprinted polymers in silica pores (MIP@C-dots) in situ. Removed the original template molecules, the MIP@C-dots composite exhibits quite high selectivity for acetylthiocholine (ACh). With AChE, its substrate ACh will be hydrolyzed into thiocholine and the fluorescence signals exhibit a dramatic decrease at 465 nm, Under optimal conditions, the fluorescent probe shows sensitive responses to AChE in the range of 0.01-0.6 mU/mL. The detection limits of AChE are as low as 3 µU/mL. These experiments results validate the novel fluorescent probe based on MIP@C-dots composite, paving a new way to evaluation of AChE activity and Screening inhibitors.

Effects of core size and PEG coating layer of iron oxide nanoparticles on the distribution and metabolism in mice.

INTRODUCTION: In vivo distribution of polyethylene glycol (PEG)ylated functional nanoparticles is vital for determining their imaging function and therapeutic efficacy in nanomedicine. However, contradictory results have been reported regarding the effect of core size and PEG surface of the nanoparticles on biodistribution. METHODS: To clarify this ambiguous understanding, using iron oxide nanoparticles (IONPs) as a model system, we investigated the effect of core size and PEG molecule weights on in vivo distribution in mice. Three PEGylated IONPs, including 14 nm IONP@PEG2,000, 14 nm IONP@PEG5,000, and 22 nm IONP@PEG5,000, were prepared with a hydrodynamic size of 26, 34, and 81 nm, respectively. The blood pharmacokinetics and tissue distribution were investigated in detail. RESULTS: The results indicated that the PEG layer, rather than core size, played a dominant role in determining the half-life time of IONPs. Specifically, increased molecular weight of the PEG layer led to a longer half-life time. These PEGylated IONPs were mainly excreted by liver clearance. While the PEG molecular layer constituted the key factor to determine the clearance ratio, core size affected the clearance rate. CONCLUSION: Complete blood count analysis and histopathology suggested excellent biocompatibility of PEGylated IONPs for future clinical trials.

Enzyme-Nanowire Mesocrystal Hybrid Materials with an Extremely High Biocatalytic Activity.

The laccase-Cu2O-nanowire mesocrystal hybrid materials were developed with a superior catalytic activity inspired by natural biocatalysis processes in living cells that highly resemble the metal ions activation and the well-organized spatial structure of the natural rough endoplasmic reticulum. The enzyme and nanobiocatalyst activities of the obtained hybrid material exhibited an approximate 10-fold and 2.2-fold increase than the free enzyme, surpassing the currently available nanobiocatalysts. The comprehensive catalytic performance of the hybrid materials has been further demonstrated using a prototype continuous-flow reactor for the bioremediation of 2,4-dichlorophenol-contaminated water, which showed a high degradation efficiency and remarkable reusability. These new highly efficient nanobiocatalysts are expected to be used for diverse applications in biotechnology, biosensing, and environmental remediation.

The selective roles of chaperone systems on over-expression of human-like collagen in recombinant Escherichia coli.

Human-like collagen (HLC) is a novel biomedical material with promising applications. Usually, insoluble HLC was formed due to over-expression. In order to improve the production of soluble HLC, the effective chaperone proteins and their mediation roles on HLC were clarified. Trigger factor (TF) pathway with low specificity and high binding affinity to nascent chains could increase soluble HLC expression; GroEL-GroES could increase the expression level of HLC by assisting the correct folding of HLC and increase mRNA level of the gene coding for HLC by enhancing mRNA stability. DnaK chaperone system did not work positively on soluble HLC due to the unbalanced ratio of DnaK:DnaJ:GrpE, especially too high GrpE significantly inhibited DnaK-mediated refolding. The production of soluble HLC with co-expression of exogenous TF and GroEL-GroES was increased by 35.3 % in comparison with the highest value 0.26 g/L reported previously.

The transport and mediation mechanisms of the common sugars in Escherichia coli.

Escherichia coli can uptake and utilize many common natural sugars to form biomass or valuable target bio-products. Carbon catabolite repression (CCR) will occur and hamper the efficient production of bio-products if E. coli strains are cultivated in a mixture of sugars containing some preferred sugar, such as glucose. Understanding the transport and metabolism mechanisms of the common and inexpensive sugars in E. coli is important for further improving the efficiency of sugar bioconversion and for reducing industrial fermentation costs using the methods of metabolic engineering, synthetic biology and systems biology. In this review, the transport and mediation mechanisms of glucose, fructose, sucrose, xylose and arabinose are discussed and summarized, and the hierarchical utilization principles of these sugars are elucidated.

Preparation of a low-cost minimal medium for engineered Escherichia coli with high yield of human-like collagen II.

In order to reduce the production cost of human-like collagen (HLC), a minimal medium was introduced. On the base of Design of experiments (DOE), especially Plackett-Burman design and central composite design, a modified minimal medium that could give a high yield of HLC was developed. The optimum minimal medium for engineered E. coli BL21 ΔptsG contained 6.11g/L of glucose, 5.82g/L of (NH(4))(2)SO(4), 1.80´10(-4)g/L of thiamine and 3.00´10(-2)L of trace element solution, the other ingredients were same to that in M9 medium. And the HLC production of ptsG mutant reached to 0.26g/L in this optimized minimal medium, which approached to 0.27g/L produced by the strain without deleting ptsG gene in an optimized complex medium.