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The 2012 International Conference on Applied Biotechnology (ICAB 2012) was held in Tianjin, China on October 18-19, 2012. It provides not only a platform for domestic and foreign researchers to exchange their ideas and experiences with the application-oriented research of biotechnology, but also an opportunity to promote the development and prosperity of the biotechnology industry. The proceedings of ICAB 2012 mainly focus on the world's latest scientific research and techniques in applied biotechnology, including Industrial Microbial Technology, Food Biotechnology, Pharmaceutical Biotechnology, Environmental Biotechnology, Marine Biotechnology, Agricultural Biotechnology, Biological Materials and Bio-energy Technology, Advances in Biotechnology, and Future Trends in Biotechnology. These proceedings are intended for scientists and researchers engaging in applied biotechnology. Professor Pingkai Ouyang is the President of the Nanjing University of Technology, China. Professor Tongcun Zhang is the Director of the Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education at the College of Bioengineering, Tianjin University of Science and Technology, China. Dr. Samuel Kaplan is a Professor at the Department of Microbiology & Molecular Genetics at the University of Texas at Houston Medical School, Houston, Texas, USA. Dr. Bill Skarnes is a Professor at Wellcome Trust Sanger Institute, United Kingdom.



Pharmaceutical Biotechnology


Chapter 66. Promising Biomarkers: MicroRNAs at Diagnosis, Therapy and Prognostic Evaluation of Breast Cancer

MicroRNAs (miRNAs) are small noncoding RNAs with regulatory functions, which play an important role in malignancies. An increasing amount of experimental evidence has shown that many miRNAs are aberrantly expressed in breast cancer and influence breast cancer behavior and progression. Furthermore, miRNAs can act either as tumor suppressors or as oncogenes, depending on the targets they regulate, and measurements of miRNAs expression in breast cancer have diagnostic and prognostic implications. Thus, this implies that miRNAs have huge potential as biomarkers. In addition, their extreme stability and ease of detection further support the idea that miRNAs have great potential to evolve into effective biomarkers in the clinic. The objective of this review is to update current realization regarding that miRNAs are promising candidates at diagnostic, therapeutic and prognostic evaluation aspects of clinical application.

Dalin Lu, Nan Wang, Xinghua Liao, Xuan Huang, Jianhua Zhang, Zhenyu Wang, Lian Duan, Jiajie Liu, Baoshu Jin, Yue Wang, Tong-Cun Zhang

Chapter 67. Dimerization of Chemokine Receptors and its Novel Roles in Drug Discovery

G-protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins and mediate most of the transmembrane signal transduction. Approximately 50 % of all marketed drugs target GPCRs, which makes this protein family the most important drug targets. Chemokine receptors belong to GPCRs, which were perceived as monomers decades ago. However, recently there are growing evidences indicating that most of GPCRs can form dimers or higher order oligomers. Some chemokine receptors are also found existing as homodimers or heterodimers. A large number of studies have suggested that homodimers or heterodimers may exhibit specific functions, which are different from their monomeric counterparts. Meanwhile, the appearance of dimers with new signaling properties gives new chance in the search for novel drug targets. In this review, we will mainly summarize the current knowledge of the dimerization of chemmokine receptors and its potential roles in drug discovery.

Mingqing Wang, Baosheng Ge, Fang Huang

Chapter 68. The Mechanism of Apoptosis in Adenocarcinoma of Lung Cancer A549 Cells Induced by Albumin-Derived from Peanut

Peanut albumin which possesses protease inhibitor activity could be used as an anticancer regent. This paper would discuss the effect and mechanism of apoptosis induced by peanut albumin on human adenocarcinoma lung cancer A549. MTT assay was used to evaluate the inhibition of peanut albumin on 11 kinds of cancer cells. Compared with other cells, there was the great significant change in A549 cells. Morphological structures were detected by fluorescence microscope, and cell cycle was measured by flow cytometry (FCM). Annexin V/PI was used to detect cell cycle of A549 treated with peanut albumin. The differential expressions between the group treated with peanut albumin and the control were detected by Western-blot and quantitative-PCR. It was found that peanut albumin could inhibit proliferation of A549 cells, and induce apoptosis of A549. The possibility apoptosis of A549 cells induced by peanut albumin maybe correlated with mitochondrial apoptosis pathway.

Minhui Long, Yuejun Sun, Ni Chen, Zimin Zhang, Wen Chu, Yue Ma, Shenheng Luo, Zhongpeng Zhao, Aipo Diao

Chapter 69. Serum miR-124 and TNF-α are Biomarkers of Ischemic Cerebrovascular Disease

MicroRNAs are small non-coding RNAs with 19-22 nucleotides. MiR-124 expresses abundantly in brain tissue and is involved in the development of ischemic cerebrovascular disease (ICVD). Tumor necrosis factor-alpha (TNF-α) is one of the important factors of the inflammatory reaction after cerebral ischemia. To explore the roles of miR-124 and TNF-α in the process of ICVD, here, we collected the serum of 15 patients with ICVD and 5 normal individuals. The levels of miR-124 and TNF-α were detected by real-time PCR and ELISA. The results showed that compared with the normal individuals, the levels of miR-124 and TNF-α in serum were upregulated in the patients, while they were downregulated after clinical treatment, suggesting that these two factors could be used as good biomarkers for the diagnosis and prognosis of ICVD. Besides, our research also provided a theory foundation for understanding the mechanism of inflammatory response in cerebral ischemia mediated by miR-124.

Jiajie Liu, Xinghua Liao, Nan Wang, Jun Zhou, Lian Duan, Dalin Lu, Zhipeng Liu, Tingbao Yan, Deyun Ma, Xiumei Dong, Xueguang Sun, Tong-Cun Zhang

Chapter 70. A New Process for Preparation of Hydroxytyrosol

Hydroxytyrosol is one of the major natural phenolic compounds, mainly in virgin olive oil and the wastewater generated from olive oil processing. Based on its strong antioxidation, hydroxytyrosol is widely used in medical treatment, food, beauty, and other industries. Its diverse biological activities have for a long time attracted considerable interest of synthetic and pharmacological areas. In this paper, the new process of hydroxytyrosol synthesis has been explored, which starts from catechol protection, followed by Friedel–Crafts reaction with ethyl oxalyl monochloride, then the Wolff-Kishner-Huang reaction and reduction reaction with LAH, and finally deprotection. The target compound was obtained by five-step reaction with an overall yield of 23 %. The title compound was characterized on the basis of


H NMR spectral.

Yinghao Gao, Xijuan Liang, Yuanmou Chen, Fei Hu, Weizhu Liu, Peng Yu, Erbing Hua

Chapter 71. Design and Synthesis of 2-Arylbenzimidazole Analogues as Novel SIRT1 Activators for the Treatment of Type II Diabetes



-dependent sirtuin deacetylase, has emerged as potential therapeutic target for treatment of human illnesses such as type II diabetes, cancer, cardiovascular and neurodegenerative diseases. Resveratrol, a naturally occurring small molecule activator of SIRT1, has been demonstrated to improve metabolism and glucose tolerance. SRT1720, an imidazothiazole derivative, recently made as the most potent SIRT1 activator is structurally unrelated to resveratrol. In this work, we design and synthesize a series of compounds as novel potential SIRT1 activators through a two-step convenient synthetic procedure. Fourteen 2-Arylbenzimidazole analogues were characterized on the basis of


H NMR spectra. Tests for biological activity of these compounds are underway.

Fei Hu, Yuanmou Chen, Yinghao Gao, Shaolong Jia, Weizhu Liu, Peng Yu, Erbing Hua

Chapter 72. Design and Synthesis of SRT1 Activators for Potential Lead Compounds of Treatment of Diabetes

Sirtuins catalyze NAD


-dependent protein deacetylation and are key regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1–7), and SIRT1 has been proven as a key mediator of the pathways downstream of calorie restriction that has been shown to delay the onset and the incidence of age-related diseases such as type II diabetes. Increasing SIRT1 activity, either by transgenic over expression of the SIRT1 gene in mice or by pharmacological activation by small molecule activator SRT1720, has shown beneficial effects in rodent models of type II diabetes. In this paper, several small molecules were designed and synthesized through a convenient synthetic procedure. Ten newly synthesized compounds were characterized on the basis of



Weizhu Liu, Qiuyue Wang, Fei Hu, Yinghao Gao, Yingying Wang, Peng Yu, Erbing Hua

Chapter 73. Synthesis of 1,3-benzodioxol-5-ethanol and Its Derivatives

It has been reported that resveratrol can enhance Sirt1 expression and has the activation effect on the proteins related to insulin signaling. It can also ameliorate insulin tolerance in order to achieve the function of preventing and curing diabetes. In this work, we designed a new series of analogs related to the structure of resveratrol such as 1,3-benzodioxol-5-ethanol, which is synthesized from 1,3-benzodioxole by the reactions of Friedel-Crafts reaction, Wolff-Kishner-Huang and reduction. In addition, seven new 1,3-benzodioxol-5-ethanol derivatives were obtained through esterification with different substituents of organic acid. All compounds were characterized by


H-NMR. Yields of derivatives are from 13.2 to 78.8 %.

Na Ji, Yinghao Gao, Yuanmou Chen, Shaolong Jia, Fei Hu, Peng Yu, Erbing Hua

Chapter 74. Tetrandrine Inhibits Proteasomal Chymotrypsin-Like Activity and Induces Apoptosis in Human PC-3 Cells

The ubiquitin-proteasome pathway has been accepted as a promising target for cancer therapy, as it plays a vital role in cell cycle, function, and survival. In this research,

in silico

study by molecular docking predicted that Tetrandrine(TET), a compound from the roots of

Stephenia tetrandra S

Moore, could bind to the active site of the proteasome β5 subunit. In vitro assay proved that TET inhibits the chymotrypsin-like (CT) activity of purified human 20S proteasome in a dose-dependent manner with IC


of 0.8 μmol/L. After exposed to TET for 24 h, the CT activity in human prostate cancer PC-3 cells was inhibited with IC


of 35 μmol/L, and the ubiquited proteins were accumulated in dose-dependent manner. This inhibition was further proved by the accumulation of GFP-CL proteins, which is an indicator of proteasome inhibition in pEGFP-CL1 transferred PC-3 cells. The proteasomal activity inhibition by TET leads to apoptosis like morphological changes and activation of caspase-3 activity in dose-dependent manner in PC-3 cells. Based on these results, we suppose that TET is a proteasome inhibitor and induces cell apoptosis.

Li Zhang, Wanxin Shi, Weihua Cao, Xiangru Liang, Yufu Hu, Mo Chen, Guoqing Shi

Chapter 75. The Purifying Effect of Apocynum Venetum Seedlings on Estuarine Water

The water in Bohai Bay has become more and more eutrophic, causing a frequent red tide bloom. How to decrease eutrophication of this sea area is a pressing problem. The seawater often enters the estuary by flood tide, so the estuary may be a good place to remove eutrophic compounds from the seawater. This paper reported apocynum venetum seedlings growth status in water from Beitang estuary and its effects on nutrient removal. The results showed that apocynum venetum seedlings could grow both in flood tide water and in ebb tide water from Beitang estuarine, and the content of nitrogen and phosphorus in the water was significantly decreased by apocynum venetum seedlings, and the decrease level was largest for nitrite nitrogen in flood tide water. These results indicated that the eutrophic water in Bohai Bay was probably purified by apocynum venetum growing in ecological floating boat at estuarine.

Yangcang Xu, Tao Yu, Yunshan Zhong, Xiaoyan Wang

Chapter 76. Expression, Purification, and Activity Assay of Chicken Interferon-Alpha

In this paper, Chicken alpha interferon (IFN-α) gene was cloned into pHis-NusA expression vector, then the recombinant expression vector was transformed into host bacteria

E. coli

BL21. The recombinant chicken IFN-α was induced to express by IPTG, then the protein expression was analyzed with SDS-PAGE. Under the condition that the recombinant protein was induced to express with 0.1 mM IPTG at 16 °C, the expressed protein was soluble. Recombinant Chicken IFN-α was purified by Ni-metal chelate affinity chromatography. The expressed protein was shown to inhibit the replication of Hand foot and mouth disease virus in vero cells.

Yue Ma, Minhui Long, Aipo Diao

Chapter 77. Synthesis and Controlled Release of 5-Fluorouracil from Hydroxyethylchitosan: Based Polymer Prodrug

A novel water-soluble hydroxyethylchitosan-based polymer prodrug of 5-fluorouracil ((HECS-5-Fu) was synthesized through acetyl spacer via ester bond. The conjugated content of 5-Fu was 12.2 % (w). In phosphate buffer solution (pH 7.4) at 37 °C, HECS-5-Fu slowly hydrolyzed to release 5-fluorouracil-1-yl-acetic acid as characterized by RP-HPLC and LC–MS. The controlled release of drug from HECS-5-Fu powder sustained for more than 25 days. The in vitro experiment indicated that 5-fluorouracil-1-yl-acetic acid at concentration of 400 ug/mL inhibited the proliferation of about 40.0 % mouse fibroblast cell line L929. HECS-5-Fu could have a potential application as an antimetabolic material in glaucoma filtration device implantation.

Yanfei Peng, Wanshun Liu, Baoqin Han, Ruixue Zhou

Chapter 78. Telomerase is Significant as an Early Diagnostic Marker and Therapeutic Target

Breast cancer, a hormone-dependence disease, is the most common malignant tumor in women. Breast cancer incidence rates increase with age. Telomerase is a ribonucleoprotein that maintains chromosome ends and endows cells with unlimited proliferative potential. Activation of telomerase maintains a relatively stable telomere length, confers immortality on some cells, and may even lead to cancer. It has been reported that, in the tissues of the breast, the activity of telomerase can be detected in more than 90 % of invasive breast cancers, while not in adjacent normal tissues. Thus, telomerase activation plays a critical role in development of breast cancer. Telomerase has been proposed as a biomarker with diagnostic and prognostic potential in breast cancer as well as a basis for the targeted therapy of breast cancer.

Lian Duan, Nan Wang, Xinghua Liao, Jun Zhou, Dalin lu, Jiajie Liu, Xueguang Sun, Tong-Cun Zhang

Chapter 79. Isolation, Identification, Antibacterial Effects of Antibiotic Drugs, and Chinese Herbal Extracts to the Pathogenic Bacteria of Swollen Abdomen from Scophthalmus maximus in Vitro

The pathogenic bacteria was isolated from Turbot (

Scophthalmus maximu

s) with swollen abdomen, and proved to be the pathogenic bacteria by the infection experiment. In this research, VITEK-2 compact system was used to identify the pathogenic bacteria, and test the antibacterial effects of antibiotic drugs, paper diffusion method was used to evaluate the antibacterial effects of three kinds of Chinese herbal extracts on the pathogenic bacteria of swollen abdomen from Turbot in Vitro.The results showed that the pathogenic bacteria of swollen abdomen from Turbot was

stenotrophomonas maltophilia

, and the separation purity was 99 %. Without Trimethoprim,

S. maltophilia

had no CLSI salient point to the antibiotics of Ampicillin, Amikacin, Aztreonam, Cefazolin, Cefepime, Cefuroxime, Cefuroxime Axetil, Cefotetan, Ceftazidime, Ceftriaxone, Ciprofloxacin, Piperacillin, Imipenem, Gentamicin, Levofloxacin, Nitrofurantoin, Sulbactam, Tazobactam. Extract of the scutellaria and honeysuckle had different antibacterial effects to the

S. maltophilia

, and then the best bacteriostatic (MIC) and bactericidal (MBC) effects were 3.125 mg/mL and 6.25 mg/mL, respectively. However, the best MIC and MBC effects to extract from polygonum cuspidate were 100 mg/mL and 200 mg/mL, MIC and MBC effects lower than that extract from the scutellaria and honeysuckle. The results can provide reference materials for the clinical use of aquaculture and aquatic animal health cultivation.

Xuan Wu, Dongqing Bai, Guoxia Zhu, Yanbin Ji, Zhichao Jia, Peng Zhou

Chapter 80. Lichen: A Potential Anticancer Officinal Resource

Many natural products, which derived from plants, animals, and microorganisms, have been isolated as bioactive compounds with great therapeutic potential for cancer, but as a fungus, lichen has long been neglected in this area. The special symbiotic form of fungi and algae and worldwide distribution even extreme habitat of lichen contribute to its biological and chemical diversity, so, lichen becomes an amazing resource for the discovery of new anticancer drugs. The aim of this review is to show the value of lichen as a potential resource of anticancer drugs, especially highlight several lichen metabolisms and their derivatives, which can show the potentials to inhibit cellular proliferation or cytotoxicity and trigger apoptosis of cancer cells. To identify the new lead-compounds from lichen and elucidate the active principles with therapeutic potential for cancer, it is essential to establish a high-throughput screening program and dedicated collaboration among lichenologists, chemists, pharmacologists, and biologists.

Meirong Ren, Xinli Wei, Feng Xu

Chapter 81. Clinical Significance of Screening Impaired Glucose Tolerance in Essential Hypertension Patients

Objective: Monitoring data of patients with essential hypertension associated with impaired glucose tolerance then provide clinical basis for the intervention of diabetes and reduce cardiovascular and cerebrovascular disease risk factors. Method: Select 158 patients without sugar metabolic abnormalities previously, whose fasting plasma glucose is <5.6 mmol/L and diagnosed as essential hypertension. Results: After OGTT, in 158 patients, there are 99 cases (62.7 %) with normal glucose tolerance, 45 patients (28.4 %) with impaired glucose tolerance, and 14 cases (8.9 %) with confirming diabetes. Conclusion: The primary hypertensive patients with impaired glucose tolerance abnormalities show a high proportion in hypertensive patients. With metabolic syndrome, patients should be monitored regularly and actively fasting glucose and after 2 h of OGTT to detect and intervene in diabetes earlier, reduce the risk factors of severe cardiovascular and cerebrovascular complications.

Jun Zhu, Peiqing Feng, Shu Guo, Xinghua Liao, Jiajie Liu, Junfang Zhang, Tingbao Yan, Yue Wang, Tong-Cun Zhang

Chapter 82. Cardiac Hypertrophy-Specific Genes are Synergistic Activated by Myocardin and CREB-binding protein (CBP) p300

Myocardin is a remarkably potent transcriptional co-activator expressed in a broad range of embryonic and adult tissues, and it can induce cardiac hypertrophy. However, it remains poorly understood about the effects of myocardin in cardiac hypertrophy. Here, we show that the p300, a histone acetyltransferase, enhanced transcriptional activity of myocardin. Conversely, class II histone deacetylases HDAC5 suppress cardiac-special genes activation by myocardin and p300. These findings point that myocardin maybe a nexus for regulation of cardiac hypertrophic-special genes expression by changes in chromatin acetylation. Our results provide novel evidence to explore the roles of myocardin and their co-factors in cardiac hypertrophy for the future studies.

Zhenyu Wang, Xuehua Zhao, Mingzhe Li, Dongsun Cao, Tong-Cun Zhang

Chapter 83. Design, Synthesis and Biological Evaluation of the Novel Antitumor Agent 2-benzyl-3, 4-dihydroisoquinolin-1(2H)-one and Its Derivatives

Tetrahydroisoquinoline and its derivatives have a wide range of biological activities, such as antitumor, antimicrobial, and analgesic properties and so on. Some tetrahydroisoquinoline alkaloid derivatives have been studied as antitumor drug molecules, but 2-benzyl-7-substituted-ethoxy]-3, 4-dihydro-1(2H)-isoquinolinone series have not been reported yet. In this paper, we would like to report the design, synthesis, and biological evaluation of the novel tetrahydroisoquinoline derivatives. Among those compounds, four new tetrahydroisoquinoline derivatives have not been reported before. All the newly synthesized compounds were characterized by


H NMR and their antitumor activities were evaluated by using HT-29, K562 and HepG2 cell line. 2-Benzyl-7-[2-(tert-butyl-4-piperazinecarboxylate) ethoxy]-3, 4-dihydro-1(2H)-isoquinolinone demonstrated good antitumor activity against K562 cancer cell with an IC


of 1.58 μM.

Jian Lv, Lei Lv, Xiaomin Zhang, Yao Zhou, Kui Lu, Yifei lu, Yuou Teng, Hua Sun, Peng Yu

Chapter 84. Using Water Miscible Ionic Liquid to Improve the Efficiency of 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by Penicillium raistrickii

Improved efficiency of steroid microbial transformation by co-solvents is generally attributed to their positive effects on the solubility of substrate in aqueous media. Ionic liquids have been widely researched as possible ‘green’ replacements for organic solvents. In this study, the effect of water miscible ionic liquids on the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by

Penicillium raistrickii ATCC 10490

was investigated. The results indicated that the striking improvement of biotransformation of substrates up to 94.46 % were obtained after addition of [BMIM]BF4 under the optimized conditions. This also resulted in morphological changes of

P. raistrickii

mycelia in the fermentation broth with or without [BMIM]BF4. Based on the above observations, we therefore conclude that [BMIM]BF4-induced changes of mycelial morphology of

P. raistrickii

have positive effects on the production of the desired product, thus most likely contributing to the observed higher yield of steroid biotransformation in addition to [BMIM]BF4 known effects on substrate solubility.

Shuhong Mao, Na Wang, Zhijiang Ge, Boyuan Hua, Yanqing Li, Fuping Lu

Chapter 85. Nuclear Receptor Property of E2F1 for Novel Anticancer Drug Discovery

E2F1, a member of transcription factor superfamily E2F, is similar to nuclear receptors such as estrogen receptor (ER) and peroxisome proliferators-activated receptors (PPARs) in structure and function. However, E2F1 plays a paradoxical role in cancer progression control. Instead of non-transcriptional function, the paradox mainly derives from E2F1 transcriptional function, which determines cell death or survival by different cofactors recruitment. This characteristic of E2F1 establishes itself as a good target for novel anticancer drug discovery and it is promising to develop appropriate small molecules with E2F1 antagonism activity or inverse agonist activity which might be even better for tumor suppression.

Ning Zhang, Jin Li, Aimin Meng

Chapter 86. MRTF-A Promotes Migration of MCF-7 Breast Cancer Cells via Transactivation of CYR61

Myocardin-related transcription factor A (MRTF-A) is a transcriptional coactivator of serum response factor (SRF). Recent studies have found that the MRTF–SRF signal pathway might play critical role in Rho-dependent cytoskeletal rearrangements and tumor metastasis. To further explore the detailed molecular mechanisms for MRTF-A-mediated cell migration, in this study, the effect of MRTF-A on the migration of mouse fibroblast NIH3T3 cells and MCF-7 human breast cancer cells was detected using a scratch wound model, and its transcriptional activity on the promoter of Cysteine-rich angiogenic inducer 61 (CYR61) was also analyzed by luciferase assay. The results showed that overexpression of MRTF-A could significantly promote the migration of NIH3T3 cells and drive the transcriptional activity of CYR61 promoter in African green monkey kidney COS-7 cells and strongly enhance upregulation of CYR61 gene. In contrast, RNA interference-mediated knockdown of MRTF-A reduced the migration ability of MCF-7 breast cancer cells. Taken together, our results suggested that MRTF-A is involved in tumor metastasis via transactivation of CYR61.

Xuegang Luo, Chunling Zhang, Wenwen Zhao, Lei Liu, Shu Guo, Zhipeng Liu, Jing Wang, Tong-Cun Zhang

Chapter 87. The Analysis of the Inhibition Effect of Cholic Acid Derivatives on the Proliferation of Breast Cancer Cells

Cancer is one of the major causes of death worldwide. Many clinical evidences showed that the incidence of colon cancer increased after the cholecystectomy, suggesting that bile acid and its derivatives might play roles in the process of tumorigenesis and its prevention. Bile acid derivative is a kind of good candidates for prodrug design, but there are still few reports of its development for anticancer drugs either domestically or internationally. In the present study, IC50 of 9 bile acid derivatives against MCF-7 human breast cancer cells was detected by MTT assay, and the safety of several bile acid derivatives with high anticancer effect were further analyzed in human umbilical vein endothelial cells. The results showed that among 9 bile acid derivatives, cholic acid n-buthyl ester had lower IC50 against cancer cells, but it did not have cytotoxicity to normal cell at low concentration, indicating that they would be a good candidate for the development of novel anticancer agents.

Xuegang Luo, Jing Wang, Xiangchao Gu, Chunling Zhang, Xiangzheng Hu, Tong-Cun Zhang

Chapter 88. Design, Synthesis and Biological Evaluation of the Novel Antitumor Agent 5-Bromobenzofuran-3(2H)-One and its Derivatives

Aurones and auronols are naturally occurring 2-benzylidenebenzofuranone-3(2H)-one derivatives. Aurones have a limited occurrence in fruits, vegetables, and bright yellow color to flowers such as cosmos and coreopsis. Aurones possess a wide range of biological activities, such as antitumor, antifungal, phytoalexin, and so on. Some aurone derivatives have been studied as antitumor drug molecules, but 2-benzylidene-5-bromobenzofuranone-3(2H)-one series have not been reported yet. In this paper, we’d like to report the design, synthesis, and biological evaluation of the novel aurone derivatives. All the newly synthesized compounds were characterized by


H NMR and their antitumor activities were evaluated by using MTT method in HT-29, K562, and HepG2 cell lines. 5-bromo-2-(4-nitrobenzylidene) benzofuran-3(2H)-one demonstrated good antitumor activity against K562 cells with an IC


of 0.37 μM.

Lei Lv, Xiaomin Zhang, Jian Lv, Yao Zhou, Weiguo Hu, Peng Yu, Hua Sun, Yuou Teng

Chapter 89. Preliminary Study on the Mechanism of Cartilage Polysaccharide Inducing H22 Cell to Engender Immunogenicity

It was discussed that the apoptosis product of H22 hepatocarcinoma cells induced by cartilage polysaccharide (CP) could activate the body immune system to antitumor. The mice’s survival time, life extension rate (LER) and livability were measured by animal experiment. Lymphocyte proliferation was determined by MTT. The immune serum titer was identified by ELISA. The tumor protein antigen was analyzed by SDS-PAGE, Western blot, 2-D, and mass spectrometry analysis (MS). Compared with the model group, there was a significant improvement on the survival time, LER, livability, thymus index, spleen index, and stimulation index in immune group. The serum had high antibody titer which was up to 1:6,400. The tumor-specific antigen was tubulin alpha chain by MS and molecular weight is 50 KDa. The apoptosis product of H22 induced by CP had immunogenicity and could activate the immune system of the mouse to antitumor.

Guoqiang Zheng, Pan Li, Anguo Teng, Jie Zheng, Wenhang Wang, Anjun Liu

Chapter 90. Design and Synthesis of Novel 20-Substituted Hydroxycamptothecin Derivatives

Camptothecin (CPT) is a potent antitumor alkaloid isolated in 1966 by M.E. Wall. Clinical use of camptothecin in cancer therapy, however, was limited by its poor water solubility. Recently, there is a growing tendency to develop some new methods for the synthesis of novel derivatives to solve the problem. In this paper, we’d like to report the design and synthesis of a novel series of 20-substituted Hydroxycamptothecin to increase the water solubility. All the five products have never been reported before. All the products and important intermediates were characterized by


HNMR and LC–MS spectra.

Shaopeng Wen, Dewu Quan, Yao Zhou, Haiyong Jia, Peng Yu, Hua Sun, Na Guo

Chapter 91. Design and Synthesis of 5-Azacytidine Analogs

5-azacitidine is a prescription injectable drug used for the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). In this paper, we would like to report a new approach to 5-azacitidine analogs from dicyanodiamine and formic acid or benzoic acid through several efficient reactions, such as cyclization reaction, Vorbrüggen coupling, and so on. Among those compounds, two azacytosine analogs and two 5-azacitidine analogs have not been reported yet. All products and important intermediates were characterized by 1HNMR and MS spectrums.

Jianbo Xing, Hua Sun, Xijuan Liang, Yuou Teng, Peng Yu, Kui Lu

Chapter 92. Stigmasterol from the Flowers of Trollius chinensis

After being extracted with 75 % ethanol,

Trollius chinensis

extract was obtained by evaporation of its supernatant under vacuum conditions and dissolved with water, followed by extraction with petroleum ether. The petroleum ether part was collected and subjected to silica gel column chromatography and separated individual. Components were further purified by several rounds of silica gel column, followed by recrystallization with methanol. In total, three compounds were isolated from the air-dried flowers of

Trollius chinensis

and identified as β-sitosterol, stigmasterol and hellebore acid based on spectral analysis, NMR experimentation. Among these three compounds, stigmasterol was found for the first time in

Trollius chinensis


Mengmeng Zhou, Min Wang, Daoqing Xu, Qin Pan

Chapter 93. Design, Synthesis and Primary Biological Evaluation of the Novel Antitumor Agent Indoline-3-One and Its Derivatives

Indolinone displays promising antitumor properties by inhibiting various kinase families. In this paper, we’d like to report the design, synthesis, and primary biological evaluation of the novel indoline-3-one and its derivatives. All the newly synthesized compounds including the novel compound 2-(4-(trifluoromethyl)benzylidene)indolin-3-one (5f) were characterized by


H NMR and their antitumor activities were evaluated by using MTT method in HT-29, K562, and HepG2 cell lines. 2-(2-nitrobenzylidene)indolin-3-one (5d) demonstrated good antitumor activity against HT-29, K562, and HepG2 with an IC


of 2.04 μM, 2.33 μM, 2.24 μM, respectively. 2-(4-(trifluoromethyl)benzylidene)indolin-3-one(5f) demonstrated good antitumor activity against K562 and HepG2 with an IC


of 2.27 μM, 3.47 μM.

Haiyong Jia, Guojun Pan, Yiqian Wang, Shaopeng Wen, Qiannan Guo, Weiguo Hu, Peng Yu, Hua Sun, Yuou Teng

Chapter 94. Research Progress on the Anti-Rheumatoid Arthritis Drugs

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease leading to synovial hyperplasia and a series of multisystem comorbidities. Permanent disability usually occurs in 20–30 % of untreated patients within 2–3 years of the disease. Therefore, it is important to diagnose the disease and initiate treatment as early as possible. The drug therapy plays an important role in maintaining a state of very low disease activity and slowing the progression of joint damage. Traditional drug therapy in RA is a pyramid approach, in which nonsteroidal anti-inflammatory drugs (NSAIDs) serve as the base of pyramid and disease-modifying antirheumatic drugs (DMARDs) are employed relatively late. But since this approach is no longer valid, many new drugs used to manage RA have been developed. In this review, we specifically summarize the current drug therapies for the treatment of RA and briefly describe the etiology, pathology and pathogenesis of RA.

Peng Wang, Xuegang Luo, Chongxi Wang, Xinjia Wang, Guang Hu, Tong-Cun Zhang

Chapter 95. Design and Synthesis of 1H-2, 3-Dihydro-1-Pyrrolizinones Derivatives

Pyrrolizinone derivatives were discovered showing remarkable anti-inflammatory and analgesic activities. Based on the SAR summarized before, a series of pyrrolizinone derivatives were designed and synthesized in this paper. All the eight target compounds were characterized by


HNMR spectra, and the test of anti-inflammatory and analgesic activities is in progress.

Changhai Sun, Jing Cao

Chapter 96. Inhibition of iNOS to Protect Intermittent Hypoxia-Induced Hippocampal Neurons Impairment by Astragalus Extract in Rat

The aim of this paper is to research the protective effect of


against intermittent hypoxia-induced hippocampal neurons impairment in rat and lay the theoretical foundation for sleep apnea improvement in cognitive function by


. Male Wistar rats were divided into four groups: (1) blank control group; (2) normoxia group; (3) intermittent hypoxia group; (4)


treated intermittent hypoxia group. After 6-week treatment, the expression of iNOS was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) at mRNA level as well as by immunohistochemistry (IHC) and western blot at protein level. As a result,


reduced the expression of iNOS at mRNA and protein levels in hippocampus compared with non-treated groups (


< 0.05). In conclusion,


could protect intermittent hypoxia-induced hippocampal neurons impairment in rat.

Qiang Zhang, Wenyuan Gao, Shuli Man, Yun Zhang, Baoyuan Chen

Chapter 97. Prepared and Characterization of 12β, 15α-Dihydroxy- 16α,17-Epoxyprogesterone

The compound 12β, 15α-dihydroxy-16α, 17-epoxyprogesterone was biotransformed from 16α, 17-epoxyprogesterone by

Colletotrichum lini

AS3.4486, and its structure was characterized by




C NMR, HREI-MS and single-crystal X-ray diffraction. The crystal of the title compound belongs to monoclinic, space group


3 with


= 8.0655(8),


= 12.1955 (12),


= 18.589(2) Å,


= 90°,


= 4,


= 1828.4(3) Å




= 1.309 mg/m




= 0.092 mm




(000) = 776,


= 0.0407 and


=0.0792. X-ray analysis indicates that intermolecular hydrogen bonds O(2)–H(2)···O(5), O(3)-H(3)···O(2) are observed.

Yibo Wang, Yanbing Shen, Jiajia Ren, Jianmei Luo, Min Wang

Agricultural, Environmental, Marin Biotechnology and Bio-energy Technology


Chapter 98. Structure Elucidation of Two Triterpenoid Saponins from Leaves of Schima superba Gardn. et Champ

Two new oleanane-type triterpenoid saponins with two angeloyls at C-21, C-22, and a branch of tetrasaccharide moiety (Rha, Cal, Glc, GlcA, or Rha, Xyl, Glc, GlcA) at C-3 were isolated from leaves of



Gardn. et Champ. Their structures were established using one- and two-dimensional NMR and high resolution electrospray ionization mass spectrometry as 21,22-di-














-glucuronopyranoside, named schimasuoside A(


), 21,22-di-














-glucuronopyranoside, named schimasuoside B (


). There are strong antifungal effect of their mixture on

Magnaporthe oryzae


Guanghua Huo, Changling Zhang, Yingjun Zhang

Chapter 99. Study on Extraction of Xylan from Bamboo Shoot Shell

Using bamboo shoot shell as raw material, the experiment utilized the method of alkali extraction after bamboo shoot shell was pretreated. Effects of alkali concentration,extraction temperature, ratio of solid to liquid, and extraction time on yield of xylan were analyzed. The conditions of alkali extraction were: alkali concentration 5 %, ratio of solid to liquid 1:10 (g:mL),extraction time 3 h, extraction temperature 120 °C. The yield of xylan from bamboo shoot shell reached 24.77 %,which shows that:bamboo shoot shell is a good raw material with the content of 35.08 % hemicellulose in it.

Nengfu Yu, Nengliang Wu, Yu Wang, Yegou Tu

Chapter 100. Study on Preparation of Low Alcoholic Wine from Tomato

Tomato is the most popular fruit in the world and the largest tomato growing field is in China. In the present study, tomato juice was used as raw material, which is available in the region were selected for wine production. The conditions for inoculums concentration, fermentation temperature, and initial sugar degree were optimized. The results showed that the tomato juices were similar to grape juice in terms of sugar and acidity. After fermentation, the ethanol concentration was 6–7 % w/v. Enzymatic hydrolysis and fermentation conditions were optimized for producing tomato low alcohol wine. The results demonstrated that the pectase amount 2 %, temperature 50 °C, and enzymolysis period 4 h, initial sugar content 15 %, inoculums size of yeast 5 %, temperature 22 °C, fermentation period 7 days, and clarified agents amount 5 mL. The alcohol content of tomato wine was 6-7 degrees with similar sugar content and acidity of the tomato juice. The product was clear, no precipitation, and in good flavor.

Kai-ye Deng, Er-na Li

Chapter 101. Effects of Glucose Assimilation on Lutein and Chlorophyll Biosyntheses in the Green Alga Chlorella pyrenoidosa

Glucose assimilation caused a significant decrease of lutein and chlorophyll accumulation in

Chlorella pyrenoidosa

cultivated in low nitrogen medium, which are in line with the previously reported glucose-bleaching effect on the green alga

Chlorella protothecoides

cultivated in nitrogen deficient medium. Our study showed that an excessive lipid production induced by nitrogen deficiency was expected to be responsible for the decrease of lutein and chlorophyll accumulation in

C. pyrenoidosa

under heterotrophic conditions. When

C. pyrenoidosa

was cultivated in high nitrogen medium, the lutein and chlorophyll biosyntheses acted very differently: lutein content still decreased markedly whereas chlorophyll content remained stable. In addition, when we inhibited chlorophyll biosynthesis with levulinic acid, the chlorophyll a (chl a) and chlorophyll b (chl b) contents decreased markedly, but the lutein biosynthesis was not influenced at all. It was concluded that lutein and chlorophyll biosyntheses were possibly not coordinated in

C. pyrenoidosa

under heterotrophic conditions.

Tao Li, Yi-han Liu, Fu-ping Lu, Yue Jiang

Chapter 102. RNA Interference and Applications in Plants

RNA interference (RNAi) inhibits gene expression in a very specific way. It has a vital significance in the regulation of gene expression, the defense of viral infection, the controlling of jumping genes. In recent years, RNAi has become an important method of studying gene function with its high specificity, efficiency, hereditability and other significant advantages and has been used in the study of functional genomics and traits improvement. This paper simply introduces the RNAi mechanism and discusses the applications of RNAi in plant functional genomics, crop quality improvement, and resistance of disease and insect.

Yunrong An, Zhongyou Pei, Nan Xin, Haifeng Wang

Chapter 103. Isolation, Identification and Degradation Characteristics of Three Thick Oil Degrading Bacteria Strains

In order to find the microbe suited to microbial enhanced oil recovery (MEOR), three bacterial strains DS1, DS2, and DS3 with excellent degrading effect on thick oil were isolated from the thick oil and water reservoir of oil well 51-82# in Erlian Oil Field. The three bacteria were identified as Acinetobacter sp., Enterococcus sp. and Brevibacterium sp. through 16S rDNA sequence similarity search. Viscosity reducing experiment was conducted using thick oil with a viscosity of 1746 mPa·s (50 °C) from Erlian Oil Field as the substrate with the three isolated bacterium alone and their consortium. The viscosity reduced 49.1, 46.6, and 49.0 % by DS1, DS2, and DS3, respectively, during 7 days degradation experiment. However, the reduction rate of viscosity reached 57.0 % with the consortium, which was significantly higher than that of single strain. It demonstrated that viscosity reduced effect was better with the consortium of the three isolated bacteria. The consortium can be applied in MEOR.

Chan Tian, Shengyan Tian, Xianbin Liu, Lulu Qin

Chapter 104. Effect of Anti-Nematode Preparations on Physiological Traits of Cucumber Leaves Affected by Root-Knot Nematode

This paper was to study the effects of anti-nematode preparations with different mechanisms on changes of enzyme systems and membrane permeability of cucumber leaves, so as to provide reference basis for effective control of soil root-knot nematode in greenhouse. With cucumber seedlings affected by root-knot nematode as material, changes of superoxide dismutase (SOD), peroxidase (POD), relative conductivity in cucumber were tested after the seedling soil was treated by preparations of Wuxianmei, Hailvsu, Duxiandna, and Avermectin. After treated by different preparations, SOD and POD activity of cucumber leaves were higher than control, and that treated by Wuxianmei was the highest. In addition to Duxiandna, the relative conductivity content of other treatments were significantly lower than control. When cucumbers were planted for 70 days, the effect of Avermectin against root-knot nematode was the best of 64.7 %. After cucumbers were infected by root-knot nematode, different preparation treatments all had certain control effect, which made the physical indicators of cucumber have obvious change.

Shuchang Lu

Chapter 105. Enzymolysis and Microbial Transformation of Geniposide in Gardenia Jasminoides into Genipin by Aspergillus niger

A filamentous fungi strain,

Aspergillus niger

, producing β-glucosidase was screened to transform geniposide in traditional Chinese medicine Gardenia jasminoides into genipin. The conversion rate of gardenia hydrolyzed by β-glucosidase which had been extracted from fermentation broth of

A. niger

could reach 15 % at the enzyme concentration of 5 %, enzyme digestion time of 40 min, temperature at 50 °C. On the other hand, genipin was achieved with the method of microbial cell transformation. The optimum conversion conditions by cells were: β-glucosidase activity dose 10.12 U/mL at a fermentation time of 96 h, rotation speed of 180 r/min, medium capacity of 50 mL and gardenia concentration of 10 %. By HPLC analysis, the maximum conversion efficiency of 22 % was achieved. By this way, conversion efficiency was increased by 7 % and the extraction process was saved compared with enzymatic hydrolysis method.

Yu Li, Wenbin Jin, Wei Jing, Mengcheng Yao, Yanyang Tang, Fuping Lu

Chapter 106. Study on the Application of a Thermotolerant Saccharomyces cerevisiae in the Production of Bioethanol

Bioethanol has attracted extensive attention because of its advantages response to energy crisis and environment pollution. However, the ethanol production could be decreased due to the high temperature created by the exothermic reactions during the fermentation process of bioethanol. In this study, the thermotolerant

Saccharomyces cerevisiae

strain constructed in our precious work was applied to the very high gravity fermentation (VHG) process for the production of bio-ethanol. The ethanol production of F3 and the control strain AY12 via different fermentation techniques were tested. Our results illustrate that the F3 strain exhibits higher ethanol production compared to the strain AY12 in different temperatures, and that the fermentation technique of simultaneous saccharification and fermentation (SSF) possesses the predominance among the four. The orthogonal test was also performed to optimize the fermentation conditions. Finally, the ratio of nutriment and water 1:2.6, inoculum size 25 ml, fermentation period 56 h were identified as the optimal fermentation conditions and the ethanol production was 12.2 % (v/v).

Yueqiang Li, Yefu Chen, Jian Dong, Xinxin Zhang, Tong Shen, Dongguang Xiao

Chapter 107. Different Chemicals Stimulate Diapause Termination of Artemia Embryos

This paper studied the effect of the chemicals on the diapause termination of the


cysts, including reactive oxygen species (ROS) producing reagent CaO


, reactive nitric species (RNS) producing reagent NO, and antioxidant reagents tea polyphenol (TP) and ascorbic acid (VC). The results showed that all the tested chemicals could significantly promote


cysts development, and the optimum range for CaO


, TP, VC, and sodium nitroprusside (SNP, NO generators) was 38–94, 50–150, 625–2500, and 25–100 mg/L, respectively. The hatching percentage (H %) was raised from 20 % for the control group to 86 % for the CaO


treated group, 86 % for the TP treated group, 85 % for the VC treated group, and 67 % for the SNP treated group, respectively. An analysis of trehalose consumption and glycerol accumulation of the


cysts was also performed during incubation.

Yuqing Chen, Bo Zhang

Chapter 108. Dominant Bacteria TCCC15005 Used for Treatment of Alkaline Wastewater from Oil Refinery in a SBR

Biological treatment was an important and integral part of any wastewater treatment plant that treats wastewater from either municipality or industry. In this study, dominant bacteria TCCC15005 was used for treating alkaline wastewater from oil refinery in a SBR system. Subsequently, the optimized treatment conditions were investigated. The result indicated that the optimized treatment conditions were 0.5 % corn mill, 1.0 % corn steep liquor, 0.2 % MgSO4·7H


O, 1.03 g/L inoculation amount, 35 °C, pH 7.5. And the treatment time was 28 h. Under the optimal conditions, the removal rate of COD can reach 84.16 %.

Jing Yang, Hua Zhao, Xi Wang, Xin Feng, Xinhua Wang

Chapter 109. Isolation and Characterization of a New Moderately Halophilic Bacterium Strain SM. 200-5 from Solar Saltern Ponds

A new moderately halophilic bacterium strain SM. 200-5 was isolated from the solar saltern ponds with salinity 200 in Hangu Saltworks, Tianjin, China. The cells of the strain SM. 200-5 were rods and Gram-negative. They could grow in a salinity range of 30–150 and initial pH 6–11, with optimum of salinity 100 and initial pH 7. 16S rDNA alignment showed that the strain SM. 200-5 had 93–95 % similarity with those sequences of genus


sp.. Biochemical characterization analysis via API 20E system indicated that biochemical characters of the strain SM. 200-5 were partially different from


sp. ISL-25, which was its closest member in the phylogenetic tree. Therefore, the strain SM. 200-5 proposed a new species of



Gaochao Xu, Yuangao Deng, Donghui Song, Liying Sui

Chapter 110. Preparation of Wetting Powder for Biocontrol Bacillus Subtilis

Bacillus subtilis

is considered as one kind of beneficial strains which can effectively inhibit the pathogenic fungus of the plants and promote plant growth.



wetting powder exhibited better prevention and cure of powdery mildew, phytophthora blight, gray mold, and plant growth promoting ability.



wetting powder was formulated using diatomite and calcium carbonate as carrier, sodium dodecyl sulfate, PVA, and CMC−Na as co-formulants, soluble starch, sucrose, and sodium glutamate as protective agent. Optimal formulation recipe and processing technology of

B. subtilis

wetting powder were developed. The best composition was diatomite 5 %, calcium carbonate 5 %, SDS 5 %, CMC-Na 5 %, soluble starch 0.5 %, sucrose 0.5 %, and sodium glutamate 0.5 %. Quality index analysis revealed that the wetting powder reached the biological pesticide standards.

Fang Chen, Shangjing Guo, Haiying Shi, Deduo Han, Yuanjun Kang, Yu Zheng, Min Wang

Chapter 111. Isolation and Characterization of s-Butanol Tolerant Microorganisms

s-butanol can be produced indirectly from biomass by fermentation. To construct recombinant strains producing s-butanol directly, microbes naturally with high tolerance to s-butanol are required for metabolic engineering. In this study, totally 30 s-butanol tolerant microorganisms were isolated from different environmental soil samples by screening and enrichment method. Among the isolates, strains S-2, S-9, S-24, and S-26 were able to tolerate 15 g/L s-butanol. Additionally, s-butanol tolerance mechanisms were also studied by quantitatively analyzing degradation ability of the isolates. The results indicated that strains S-9 and S-24 possessed relatively low levels of s-butanol degrading enzyme activity and might be used as native tolerant strains for metabolic engineering. Strain S-13 showed the highest s-butanol degradation efficiency (72.7 %) in LB medium, suggesting that strain S-13 disrupted s-butanol via a way of co-metabolism and suitable for bioremediation of the chemical under more complicated environments. Furthermore, the isolated strains were identified by comparative analysis of the 16S rRNA gene sequences.

Litao Shi, Hongjiang Yang, Qian Li, Xuying Qin

Chapter 112. Application of Support Vector Machine in Base Liquor Classification

Considering the deficiency of the traditional liquor classification method, a novel method for liquor classification based on support vector machine is discussed in this paper. Liquor chromatographic data is used as basis and the LIBSVM toolbox is used as classification tool in this method. Two different grades of 490 base liquor samples (containing 242 samples of ordinary base liquor, 248 samples of high-quality base liquor) were used to test the method. In the experiment, 180 samples of ordinary base liquor and 184 samples of high-quality base liquor were selected as the training set to build the model and the remaining base liquor were used as the testing set to test the accuracy of the model. The model accuracy could reach 98 % without the correlation parameter optimization. The results show that the method can achieve a higher accuracy, and prove the correctness and effectiveness of the method.

Junsen Lu, Liping Du, Haimei Ding, Ziping Du, Dongguang Xiao

Chapter 113. Isolation and Characterization of n-Butanol Tolerant Microorganisms


-butanol is produced traditionally by acetone butanol ethanol (ABE) fermentation with relatively low productivity. To improve n-butanol synthesis level, microbes naturally with high tolerance to n-butanol are required for metabolic engineering. In this study, a total of 30 n-butanol tolerant microorganisms were isolated from various environmental samples by screening the enriched cultures. Ten of them were able to tolerate more than 2 % (v/v) n-butanol, and strain YY-23 even thrived in the presence of 3 % (v/v) n-butanol. In addition, n-butanol tolerance mechanisms were investigated by quantitatively analyzing degradation ability of the isolates. The results showed that strains YY-13, YY-14, YY-15, and YY-18 didn’t metabolize n-butanol in LB medium, suggesting that they didn’t have n-butanol degrading enzymes and all of them might be used as candidates for construction of high n-butanol producing strains. Strains YY-10 and YY-29 showed strong n-butanol degradation efficiency (31 and 20 %, respectively), and might be used for n-butanol bioremediation. Furthermore, the isolated strains were identified by the 16S rRNA gene sequences comparative analysis.

Yue Yu, Hongjiang Yang, Qun Li, Xuying Qin

Chapter 114. Effects of Dual-Frequency Ultrasound with α-amylase on the Properties and Structure of Mung Bean Starch

In this paper, we focus on the effects of dual-frequency ultrasound (40 and 80 kHz) with α-amylases (5U/g) on the properties and structure of mung bean starch. The reducing sugar value and starch solubility in the treated starch slurry under ultrasound enzyme were studied. Scanning electron microscopy (SEM) and Fourier transform infrared spectrometer (FTIR) were used to measure the apparent structure and crystal texture of starch. The results indicated that compared with the results of single-frequency ultrasound treatment, dual-frequency ultrasound promoted the starch hydrolysis by amylase and increased the starch solubility remarkably. More channels and holes were found on the surface of treated starch granule by SEM. The results of FTIR analysis showed the FTIR peak shape of ultrasonic treated starch was different from that of the control starch.

Aijun Hu, Jing Lu, Jie Zheng, Xiaoqing Zhang, Ying Zhang, Tong-Cun Zhang, Qian Li, Lin Yang

Chapter 115. Relationship Between Diet and Stable Carbon and Nitrogen Isotope Composition in Beef Tissues

The effect of the amount of C


plants in diets on the carbon and nitrogen stable isotope composition of different types of beef tissues was investigated. Eighteen young bulls were randomly divided into six groups and fed with a diet consisting of different content of C


plant material. At the end of experiment, the δ


C values of cattle tail hair, defatted muscle, and crude fat all become enriched and significantly correlated with increasing proportions of C


constituents in the diet. However, the δ


N values of beef tissues did not increase regularly with the change of diet, but the correlation among δ


C and δ


N values of above tissues were highly correlated. Based on these results, it is possible for the proportion of C


plant material to be estimated from the δ


C values of the different tissue samples. And, cattle tail hair instead of muscle in beef origin is available.

Fengmei Sun, Guangyu Shi, Huiwen Wang, Shuming Yang

Chapter 116. Expression and Sequence Analysis of ChRpS3, a Ribosomal Protein S3 cDNA, and its Potential Role in Ovary Development of Cymbidium hybridium

A full length cDNA sequence,




, was isolated by RT-PCR and further characterized by sequencing and expression analysis. The cDNA fragment was 1,003 bp in length and contained a complete open reading frame of 786 bp, encoding a protein of 261 amino acid residues with high sequence homology to RpS3 from other plants such as monocotyledon

Oryza sativa

(91 %). Phylogenetic analysis at the amino acid level also confirmed that




belonged to the monocot-specific RpS3 clade. Furthermore, spatial and temporal expression analysis with RT-PCR indicated that




was highly expressed in ovaries 2 days after pollination and is predicted to encode a novel member of RPS3 participating in cell growth and proliferation. It is deduced that this 40S ribosomal S3 like protein is involved in initiating ovary development of

C. hybridium

, which might provide novel insights into the molecular mechanism of ovary and floral development.

Xiaoqiang Chen, Xiulan Li, Ning Sun, Wenqin Song

Chapter 117. Lichen Flora on the Genera Alectoria, Pseudephebe, and Sulcaria (Lichenized Ascomycota, Parmeliaceae)from the Hengduan Mountains in China (4)

Alectorioid lichen specimens used in this study were collected since 1981, mainly from the Hengduan mountain region, all the specimens were examined using standard microscopic techniques and hand-sectioned under dissecting microscope, secondary metabolites were identified by TLC. After detailed taxonomical and chemical study of our specimens and the type specimens,





is found to have same diagnostic characters with




except for the diameter of the main branch, thus, it is treated as the synonym of









, and




have rather restricted distribution in Hengduan Mountains, they are considered as rare lichens. Taxonomic characters together with distribution map and key to Alectorioid lichens are provided in this paper. All the specimens involved in this study are deposited in Herbarium of Kunming Institute of Botany (KUN).

Xinyu Wang, Dong Liu, Jianwen Li, Hiroshi Harada, Lisong Wang

Chapter 118. Discussion on the New and the Old Country Mark in Detecting the Coliform Bacteria

For the application of the new country mark in practice detection, the lactose method and the LST method were compared. Lactose method and LST method were used to determine the different consistence bacterium fluid of the coliform bacteria and pollution-free eggs, beverages and tablewares, which had 110 samples in all. The result of two different detection methods had no difference in statistics. Within the MPN 95 % confidence interval, lactose method and LST method were credible and accurate. Compared with lactose method, the unqualified rate of LST method in sample detection was increased by an average of 48.4 %. Although there was no difference between these two kinds of methods in statistics, the unqualified rate of LST method was absolutely higher than lactose method. This result showed that LST method could reduce the rate of false negatives in sample detection.

Lin Huang, Chunxia Wang, Ying Zhang, Fan Mei, Yan Huang, Jinpeng Wang, Bo Zheng

Chapter 119. Screening Autotetraploid Plantlets of Glycyrrhiza uralnesis Fisch by Colchicine-Treated Bud Culture

To screen effective and useful autopolyploid of

Glycyrrhiza uralnesis Fisch

, the buds were used to dip in the medium with colchicine at different concentrations for different times. The treated buds were transferred to pots with perlite to grow at room temperature. After 2 weeks, the root tips from the cultured seedlings were cut to observe their cell morphology and chromosome counts, and the individual contained four chromosome groups harvested as an autotetraploid. The results indicated that, the colchicine of low concentration might promote the bud′s development, whereas high concentration colchicine is harmful for the buds of

Glycyrrhiza uralnesis Fisch.

Moreover, over 30 % autotetraploids might be obtained using 0.15 or 0.20 % of colchicine for 12–36 h, but in comparison with the diploids, the plantlets of many autotetraploids became badly weak at first generation. Therefore, the autotetraploids preliminary obtained will be faced to strictly screen according to agricultural traits and effective component.

Xinglin Li, Junting Lu, Xuefei Cao, Na Zhao, Yang Han, Aijia Cao, Jie Ding, Jun Zhao

Chapter 120. Screening of Antimicrobial Marine Microorganisms and Purifying of Its Bioactive Substances

A bacterium HS-A38 with antimicrobial activities was isolated from the intestine of wild sea cucumbers in Dalian sea area. Based on the analysis of morphological, physiological, and 16S rDNA sequence, the strain HS-A38 was identified as Bacillus subtilis. Two bioactive substances (1 and 2) were purified from the fermented broth of the Bacillus subtilis HS-A38 using the methods of fractional sedimentation with ammonium sulfate, CM-52 ion-exchange chromatography and Sephadex G-75 column. The SDS-PAGE analysis indicated that the relative molecular weight of these bioactive substances were 41 kDa and 28 kDa, respectively. The antibacterial spectra showed that substance 1 could only inhibit Gram-positive bacteria, whereas substance 2 could significantly inhibit both the Gram-positive and negative bacteria.

Zhiwen Liu, Qiankun Ruan, Sirigulen Qian, Lina Cong

Chapter 121. Determination of Dichloromethane in Waste Water Using Headspace Gas Chromatography

A method for the detection of dichloromethane (DCM) in waste water was developed by using head-space gas chromatography with FID detection. The calibration curve was linear in three orders of magnitude range of DCM levels and the linear correlation coefficient was 0.9995. The limit of detection was 0.004 mg/l and the limit of quantitation was 0.01 mg/l. The system and method precision were both accord with the requirement. The method, practiced in the quantitative determination of DCM in waste water, solved many problems as such and high water content in sample. Many impurities which impacted the detection of DCM were separated visibly.

Chaozheng Zhang, Huijing Xu, Yutao Li, Fuhai Wang

Chapter 122. Simulation of Bio-syngas Production from Biomass Gasification via Pressurized Interconnected Fluidized Beds

Bio-syngas production from biomass gasification via pressurized interconnected fluidized beds was described. The interconnected fluidized beds technology separates the gasification and combustion processes of biomass, and the heat is transferred from combustor to gasifier by bed materials, while extra heat needed in gasification process is provided by additional biomass burning in the combustor. The simulation of the whole process was carried out with Aspen Plus software. The effects of gasification temperature (



), gasification pressure (



) and steam to biomass ratio (


) on bio-syngas production were studied. The results showed that gasification temperature, gasification pressure, and


had great influences on the bio-syngas composition and to achieve high carbon conversion and yield of high-quality bio-syngas, the suitable gasification temperature is around 750 °C, and the gasification pressure and


could not too high.

Fei Feng, Guohui Song, Laihong Shen, Jun Xiao

Chapter 123. Research on Salt-tolerant Gene GPD1 in Zygosaccharomyces rouxii

To better understand the osmo-adaption mechanism, salt-tolerant gene


coding for glycerol-3-phosphate dehydrogenase was evaluated in the wild-type

Zygosaccharomyces rouxii

(S). Genomic DNA of S was used for PCR reaction to amplify the


gene. The PCR products were sent into sequencing vector pUC19 to achieve the plasmid pUC19-


. The engineered strain W303-YEp195-S and the control strain W303-YEp195 were constructed, respectively. It was observed that the overexpression of S


can obviously increase the tolerance of salt compared to the control. Identification, and overexpression of S


from the S play an important role for the osmo-adaption mechanism of salt-tolerant yeasts used in soy sauce fermentation.

Lihua Hou, Yanfei Yu, Cong Wang, Chunling Wang

Chapter 124. Mutagenic Research on Ciliary Neurotrophic Factor (CNTF) in Escherichia coli After Heavy Ions Irradiation

Based on the natural human CNTF gene, Ying et al. acquired CNTF-T mutant and constructed

Escherichia coli

expressing CNTF. In order to increase the yield and enhance the protein expression in the constructed

E. coli

, the strains were irradiated with high-LET heavy ions. Some mutants were obtained in this work and the mechanisms underlying the enhanced expression of CNTF in

E. coli

after exposure to heavy ions are discussed in this paper.

Xiaodong Jin, Qingfeng Wu, Xinguo Liu, Yan Liu, Yong Chen, Jian Lu, Lin Jiang

Chapter 125. Molecular Characterization and Expression of Ribosomal Protein L15 Gene (RPL15) From Arachis hypogaea

RPL15 is a component of the large ribosomal subunit 60S. The sequences of cDNA and DNA of


were cloned successfully from



cultivar Ri Hua 1. These two sequences were analyzed preliminarily. The full length


cDNA of

A. hypogaea

contains a 615-bp open reading frame (ORF) which encodes 204 amino acids. The length of


gene was 1383 bp with three exons and two introns. Primary structure analysis revealed that the molecular weight of the putative RPL15 protein is 24211.09 Da with a theoretical pI of 11.44. The deduced amino acid sequence of RPL15 (

A. hypogaea

cultivar Ri Hua 1) shared homology with other reported species. The expression of


transcript showed that


mRNA was expressed mainly in root. The cDNA of


was cloned successfully from

A. hypogaea

in this study. It provides scientific material for enriching and improving the


gene database.

Qi Wu, Xiuzhen Wang, Hongtao Yu, Yufei Ding, Fenggao Cui, Jiancheng Zhang, Yueyi Tang, Chuantang Wang

Chapter 126. Optimization of Extraction Conditions for Glycosaminoglycan from Urechis unicinctus by Response Surface Methodology

The enzyme dosage, enzymolysis time, and alcohol precipitation concentration as independent variables and the extraction rate as the response value, the extraction technology of glycosaminoglycan from

Urechis unicinctus

was optimized via response surface methodology on the basis of single-factor experiments. The extraction rate of glycosaminoglycan was affected in the following order: concentration of alcohol precipitation > enzymolysis time > enzyme dosage. The optimal conditions for the glycosaminoglycan extraction were as follows: alcohol precipitation concentration 75 %, enzymolysis time 4 h, and enzyme dosage 0.4 %.

Chunying Yuan, Xu Han, Qingman Cui, Ping Liu

Chapter 127. Quantitative Trait Loci Mapping of Unstripped Germ Rate in Milled Rice

The objectives of this study were to investigate the genetic factors controlling unstripped germ rate in milled rice using Quantitative trait loci (QTL) analysis. A linkage map consisting of 80 DNA markers was constructed by using 224 recombinant inbred lines (RILs). A total of 8 QTLs located on chromosomes 3, 4, 6, 7, 8, 9, and 12 were detected. These QTLs explained phenotypic variations ranging from 26.44 to 57.28 %. The results and the tightly linked molecular markers that flank the QTL will be useful for improvement of quality in rice breeding.

Shengbin Liu, Fang Wang, Zetian Hua, Meng Meng, Fei Zhao, Xin Liu

Chapter 128. The Application of the GSI in the Preservation and Quality Control of Oat Beverage

The purpose of this paper is to introduce the concept of Globle stability index (GSI) in the quality control of oat beverage. The Q10 value of oat beverage was determined by protracting the GSI curve. The results showed that the Q10 were 1.40 and 3.00, respectively, when the temperatures were changed from 28 to 38 °C and 38 to 48 °C. The GSI indicator of oat beverage was established. The key factors of the quality control of oat beverage were total acid, total sugar, viscosity, soluble solids, precipitation, chromatic aberration, and pH. The GSI equation was y = 0.9889e−0.001x, R = 0.9898. The impact factors of the simplified GSI test through experiments and data processing were determined, included soluble solids, precipitation, color, and pH. The simplified GSI equation was y = 1.0021e−0.0012x, R = 0.9746. The GSI indicator of oat beverage was established through the application of the two curves. That provided technical preparation for the production of oat beverage.

Yuzhu Liu, Min Zhang

Chapter 129. The Effect of Carbon Nanotubes on Rice Seed Germination and Root Growth

There are few researches available on the effects of carbon nanotubes on rice seed germination and root growth. Aiming at this problem, rice seeds were co-cultured with carbon nanotubes of different concentrations. The rice seedling growth situation was observed including germination percentage rates, length of seeding stem and root. The absorbance was measured and the root activity was calculated. Experiment results showed that the rice seed germination and root growth were promoted by carbon nanotubes with appropriate concentrations (0 ~ 100 μg/ml). When the concentration increased to 150 μg/ml, the root length, root activity and stem length decreased in comparison with the value of 100 μg/ml, but still slightly higher than the control, although the root and stem lengths were shorter than the control. Our results demonstrated, for the first time, that carbon nanotubes could promote rice seed germination and root growth at lower concentrations, and may have toxic effects at high concentrations.

Yumei Jiang, Zetian Hua, Yiqing Zhao, Qindai Liu, Fang Wang, Qin Zhang

Chapter 130. Study on the Accumulation Laws of Protein in Japonica Rice Seed During Development

Rice protein is nutritionally superior over most of the other food cereals in terms of amino acid composition. But rice is usually a low protein crop. So, it is important to investigate the change laws of protein for breeding rice with high protein content. The rice seed start to accumulate protein after flowering. In this study, Z601, a


rice variety, was used as the experimental material. Seeds of five days, ten days, fifteen days, twenty days, and twenty-five days after flowering (DAF) were collected. Protein contents were determined by Kjeldahl method in order to confirm the surge stage in rice seed. The components of rice protein at various development stages were analyzed by SDS-PAGE analysis to find the accumulation law of the individual protein components. The results showed that 15–20 DAF was the surge stage of protein accumulation in rice seed. The protein components in the seeds of 5 DAF did not include albumin and globulin and only contained glutenin and gliadin. After that stage albumin and globulin start to synthesis and the components of protein tended to be stable. The vast majority content of protein during rice seed development was glutenin with about 48 % of the total. The proportion of albumin to total protein content is 20 % and globulin is 15 %, also as gliadin.

Fang Wang, Bolian Sun, Chunkai Gu, Jiajia Mi, Qin Zhang, Zetian Hua

Chapter 131. Prevalence Investigation of Tetracycline Resistant Bacteria in Raw Milk

To evaluate the prevalence of tetracycline resistant bacteria in raw milk, totally 198 raw milk samples were collected from Hohhot, Inner Mongolia. Preliminary screening results showed that 187 isolated strains were resistant to tetracycline. Fifteen strains were randomly selected for comparative 16S rRNA analysis, and they belonged to different genera including














(1), and one unclassified Bacillales strain, respectively. Their tetracycline resistance phenotype was further validated with K–B disk diffusion method, and 11 strains were resistant to tetracycline and 4 isolates intermediate. Tetracycline resistant determinants were investigated by PCR method. Gene




, and


were found in 11, 8, and 1 isolates, respectively. Furthermore, their hemolytic phenotypes were also characterized. The results showed that tetracycline resistant bacteria with a relatively high diversity commonly existed in raw milk with a high prevalence.

Xiaomei Zhang, Hongjiang Yang

Chapter 132. Study on Biological Denitrification Removal Technologies Treating Eutrophication Water

In this paper, efficient bacteria including photosynthetic bacteria, nitrifying bacteria, compound bacteria, etc., were applied to degrade pollutants of the urban lakes. The results showed that the removal rates of organic substances, chlorophyll-a and nitrogen compounds were 60, 90, and 50 %, respectively. Moreover, dissolved oxygen (DO) content increased from 1 to 7 mg/L and pH value remained at about eight by adding microbes. The conclusion can be drawn that it is a feasible method for adding microbes into eutrophication water for its decontamination.

Zongzheng Yang, Huan Zhang, Deqiang Zhang, Jinzhao Pang

Chapter 133. Study on Decolorization Effect of Biological Strengthening the Activated Carbon with White Rot Fungi

The colority and Chemical Oxygen Demand (COD) removal rates of biological strengthening the activated carbon with

Phanerochaete chrysosporium



for papermaking and printing-dyeing wastewater were investigated. With 1 % (v/v)

Phanerochaete chrysosporium,

the colority removal rate for papermaking wastewater reached 80 % at 3 h of retention time, and for printing-dyeing wastewater at 1.5 h of retention time; the COD removal rate was 65.14 and 78.78 % respectively at 3 h of retention time. Having been treated by 1 %


the colority removal rate was lower than that induced by

Phanerochaete chrysosporium,

and the COD removal rate was 71.35 % for papermaking wastewater. Combined strains treatment produced the colority and COD removal rate of 90 and 80 % respectively for papermaking wastewater. The COD removal rate of the biological strengthening activated carbon technique strengthened by


first was higher than that strengthened by

Phanerochaete chrysosporium


Zongzheng Yang, Junxia Xu, Peng Yu, Jinzhao Pang

Chapter 134. The Removal of Crude Oil in Waste Drilling Muds by a Constructed Microbial Consortium

Waste drilling muds (WDMs) contain serious pollutants produced by crude oil and gas well drilling. Bioremediation has been known as a useful and cost-effective method for disposal of contaminants in various environments. In this study, an efficient and stable microbial consortium was constructed successfully by successive enrichment of indigenous microorganisms, which can remove oil contaminants from waste drilling muds. Denaturing gradient gel electrophoresis analysis (DGGE) and 16S rRNA gene clone library analysis were used to evaluate the dynamic changes in the microbial consortium in the process. After 7 days’ treatment, the main contaminants were removed mostly by constructed microbial consortium than control treatment, which showed that the constructed consortium was of great potential to remediate polluted drilling fields.

Yunkang Chang, Xingbiao Wang, Yifan Han, Manman Wang, Chenggang Zheng, Yongli Wang, Zhiyong Huang

Chapter 135. Isolation and Identification of Saline Tolerance Phosphate-Solubilizing Bacteria Derived from Salt-affected Soils and Their Mechanisms of P-solubilizing

The salt-affected soils of beach from Tianjin China were sampled to screen the saline tolerance phosphate-solubilizing bacteria (SA-T-PSB) using inorganic phosphorous medium. On basis of the phenotypic characterization and 16S rRNA gene sequencing, 4 isolates with the highest PSA, B1114, B1213, B1303, and J101 were identified as

Enterobacter ludwigii,

Pantoea ananatis, Pseudomonas psychrotolerans,


Gluconobacter frateurii

, respectively. Subsequently, aimed to assaying the mechanisms of P-solubilizing, organic acid types of the 4 isolates were determined by high performance liquid chromatography. The results showed that all 4 isolates mainly secreted gluconic acid. The effect of carbon and nitrogen source on P-solubilization activity of the strains also indicated that the production of gluconic acid is the main mechanisms of P-solubilizing.

Yang Han, Chunmei Wang, Xinglin Li, Xuefei Cao, Aijia Cao, Na Zhao

Chapter 136. Isolation and Characterization of a Bacillus Strain for Alkaline Wastewater Treatment

Biological treatment is one of the considerable choices for removing of organic pollutants present in petrochemical wastewaters. In this study, five strains, named as BS1, BS2, BS3, BS4 and BS5, were isolated from sludge nearby a petroleum smelter. BS5, the isolate with the highest COD removal rate, was identified as

Bacillus flexus

, based on 16S rDNA sequences. Subsequently, the optimized COD removal conditions of BS5 were investigated. It was indicated that the optimal conditions were 0.5 % corn starch, 1 % corn steep liquor, 35 C, pH 7.5, and 10 % (v:v) inoculation size. Under such circumstance, the removal rate of COD can reach 81.04 %. The isolation of

Bacillus flexus

strain BS5 provided an alternative for the bioremediation of alkaline wastewater. Lastly, the study showed that consecutive disposal process may help to reducing COD of wastewater effectively.

Kun Chen, Wenyu Shi, Jing Yang, Tong-cun Zhang, Hua Zhao
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