Biochemical characterization of cholesterol degrading bacillus strains

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS BIOCHEMICAL CHARACTERIZATION OF CHOLESTEROL-DEGRADING BACILLUS STRAINS Student : VU THI LINH Class : K62CNSHE Student’s code : 620594 Supervisor : Assoc Prof Ph.D NGUYEN VAN GIANG Hanoi – 2022 COMMITMENT I hereby declare that the data and research results in this thesis are true and have never been used in any publication I hereby declare that any assistance in the presentation of this thesis has been received and that the sources cited have been acknowledged Hanoi, May 12, 2022 Student Vũ Thị Linh i ACKNOWLEDGEMENT During the process of completing my graduation thesis, I personally received a lot of encouragement and support, along with the enthusiastic and thoughtful guidance of teachers in the Department of Microbial Biotechnology, Biotechnology Faculty, Vietnam National University of Agriculture I would also like to express my deep gratitude to Ass Prof Dr Nguyen Van Giang, Head of Department of Microbial Biotechnology, Biotechnology Faculty, Vietnam National University of Agriculture, and Dr Truong Quang Lam, Key Laboratory for Veterinary Biotechnology, who has wholeheartedly guided and helped me throughout the process of doing my graduation thesis I also sincerely thank the professional engineers, Mrs Nguyen Thi Thu, Ms Tran Thi Dao and others at the Department of Microbial Biotechnology for creating favorable conditions for me to complete the report in the best way Finally, I would like to thank my family and friends, who have always helped and encouraged me throughout the process Sincere thanks! Hanoi, May 12, 2022 Student Vũ Thị Linh ii INDEX COMMITMENT i ACKNOWLEDGEMENT ii LIST OF TABLES .v LIST OF FIGURES v LIST OF ABBREVIATIONS vii ABSTRACT viii I INTRODUCTION 1.1 Introduction 1.2 Purpose and requirements .3 1.2.1 Purpose .3 1.3 Scientific and practical significance 1.3.1 Scientific significance 1.3.2 Practical significance II LITERATURE REVIEWS 2.1 Introduction to cholesterol 2.1.1 Cholesterol classification 2.2 An overview of Bacillus strains that could break down cholesterol 13 2.2.1 General information about Bacillus spp 13 2.2.2 Some Bacillus strains able cholesterol degrade 15 2.2.3 Determining cholesterol concentration in microorganisms 17 2.2.4 Bacillus strains that degrade cholesterol are being studied in Vietnam and across the world 18 III MATERIAL, AND METHODS 21 3.1 Materials, chemicals, and equipment 21 3.1.1 Materials 21 3.1.2 Chemicals and equipment .21 iii C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 3.2 Methods .22 3.2.1 Screening for cholesterol degrading microorganism 22 3.2.2 Characterization biochemical of selected strains 23 3.2.1.2 Endospore staining (Nguyễn Lân Dũng, Đinh Thúy Hằng, 2006) 24 3.2.3 Identification of strains L1.5 and G9.4 .26 3.2.4 Effect of different pH .27 3.2.5 Effect of different carbon sources 27 3.2.6 Effect of different nitrogen sources 27 3.2.7 Effect of different incubation times 28 3.2.8 Tolerance to Bile Salt .28 IV RESULTS 29 4.1 Screening for Bacillus strains capable of degrading cholesterol 29 4.2 Characterization biochemical of selected strains 32 4.3 Identification of strains L1.5 and G9.4 34 4.4 Effect of different pH 35 4.5 Effect of different carbon sources .37 4.6 Effect of different nitrogen sources .38 4.7 Effect of different incubation time 40 4.8 Tolerance to bile salt 41 V CONCLUSION AND SUGGESTION 42 5.1 Conclusion 42 5.2 Suggestion 42 REFERENCES 43 Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn iv C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an LIST OF TABLES Table Components of the medium 21 Table The efficiency of cholesterol degradation 31 Table Characterization biochemical of selected strains 32 Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn v C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an LIST OF FIGURES Fig Chemical structure of cholesterol Fig 2 Low density lipoproteins (LDL) .5 Fig Atherosclerosis 3d illustration Fig Cholesterol-rich plaque (yellow) builds up inside your arteries Fig Reaction catalyzed by cholesterol oxidase 13 Fig Image of B subtilis 168 obtained using a scanning electron microscope 14 Fig Process of MALDI TOF 27 Fig Cholesterol Standard Curve 29 Fig Microorganism strains capable of degrading cholesterol 29 Figure Biochemical test of selected strains 33 Fig 4 Effect of different pH to optical absorbance 36 Fig Acid tolerance of L1.5 and G9.4 strain at pH .36 Fig Effect of different carbon sources to optical absorbance .37 Fig Effect of different nitrogen sources to optical absorbance 38 Fig Effect of different incubation time to optical absorbance .40 Fig Tolerance to bile salt of strains 41 Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn vi C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an LIST OF ABBREVIATIONS WHO World Health Organization CVDs Cardiovascular diseases LDL Low-density lipoprotein HDL High-density lipoprotein HMG-CoA β-Hydroxy β-methylglutaryl-CoA SGOT Serum glutamic-oxaloacetic transaminase SGPT Serum glutamic pyruvic transaminase CHO Cholesterol oxidase HDL-C High-density lipoprotein cholesterol LDL-C Low-density lipoprotein cholesterol CHD Coronary heart disease EPA Eicosapentaenoic acid DHA Docosahexaenoic acid VLDL Very-low-density lipoprotein LCAT Lecithin cholesterol acyltransferase Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn vii C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ABSTRACT Cardiovascular disease is one of the main causes of death worldwide There are many causes of this disease, but cholesterol is one of the most related and mentioned factors Besides using drugs to reduce LDL levels, currently, there is a growing interest in new research directions involving the use of enzymes extracted from microorganisms that have the effect of lowering bad cholesterol levels Microbial strains isolated from the gastrointestinal tracts of animals were collected and screened to select those capable of breaking down cholesterol Out of twenty-six strains, two were selected (L1.5 and G9.4) that could use cholesterol as a source of nutrients Bacterial strains L1.5 and G9.4 exhibited a cholesterol degradation percentage of 55.74% and 54.63%, respectively Some parameters of pH, nitrogen source, carbon source, culture time, and bile salt tolerance of the two selected strains were evaluated through the optical density measurement method The results showed that L1.5 strain best develops at pH = 7, D-glucose, KNO3, culture time is 48 hours After 48 hours of culture in the appropriate culture medium at pH = 7, strain G9.4 grows well when no carbon source and NaNO3 are added to the medium Using MALDI TOF and biochemical characteristics: Strain L1.5 was identified as Bacillus amyloliquefaciens, whereas strain G9.4 was recognized as Bacillus flexus Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn viii C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an I INTRODUCTION 1.1 Introduction According to World Health Organization (WHO) estimates, cardiovascular diseases (CVDs) are the leading risk factor, claiming the lives of an estimated 17.9 million people each year Most CVD-related risk factors, such as nicotine use, real lethargy, diabetes, fat, and high blood pressure However, risk variables such as blood pressure and cholesterol continue to be the most commonly reported (Mc Namara et al., 2019) (Aguiar et al., 2014) Many researchers have achieved impressive clinical outcomes on the relationship between patients undergoing CVD therapy and blood cholesterol decrease (Pousinho et al., 2016) Cholesterol is an animal sterol present in vertebrates' bodily tissues Large amounts of it may be detected in the liver, spinal cord, and brain Cholesterol is a component of cell membranes that helps to keep them stable Cholesterol is produced from two sources: endogenous and exogenous Endogenous cholesterol, which is created in the liver daily in amounts ranging from 1.5 to 2g Exogenous cholesterol is created by the consumption of animal fatcontaining foods such as meat, cheese, milk, egg yolks, and animal viscera There are two main kinds of cholesterol: low-density lipoprotein (LDL), and high-density lipoprotein (HDL) High-density lipoproteins are a heterogeneous group of macromolecules with different physical properties and chemical components The function of HDL seems to be the reverse transport of cholesterol from different tissues into the liver LDL cholesterol, which transports cholesterol from organs to blood vessels, is called harmful cholesterol When LDL cholesterol levels are high in the blood, plaque forms in the arteries, leading to heart disease and stroke High cholesterol is responsible Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 4.4 Effect of different pH pH is one of the parameters influencing the mass development of bacterial cells via membrane transporter activity involved in intracellular pH regulation (Guan, N., & Liu, L 2020) The survey was done in a medium enriched with cholesterol at a pH range of to Also, at a spectrum of 650 nm, quantify the optical density of bacterial strains The effect of different pH is shown in Fig These bacteria strains can survive in a variety of pH conditions Strain G9.4 has adapted to a wide range of culture mediums pH, from acidic to neutral When the pH of the media reached 7, the most developed had an OD650 value of 0.548 Strain L1.5 grew better under neutral culture conditions, with the highest results at pH 7, when the optical density of the strain's growth reached 0.621 This strain cannot grow in a high acid media and develops slowly on a medium with a pH of Hasina Wali et al (2019) discovered that pH is a good environment for bacterial strains to develop when testing the cholesteroldegrading capacity of two Bacillus pumilus W1 and Serratia marcescens W8 strains When the two experimental strains are exposed to an environment with high acidity, their capacity to break down cholesterol is typically reduced At pH 7, all Bacillus cultures show the highest capacity to break down cholesterol, similar to the Bacillus bacteria identified from Ghee (Kokila and Amutha, 2016) Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 35 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Optical density at 650nm 0.7 G9.4 L1.5 0.6 0.5 0.4 0.3 0.2 0.1 Different pH Fig 4 Effect of different pH to optical absorbance Acid tolerance Fig Acid tolerance of L1.5 and G9.4 strain at pH The acid tolerance of both microorganisms was studied in this report According to the results (Fig 10) of the capacity to grow on LB medium with pH after hours of testing, these two strains are capable of growing on acid media Based on Figure 3, it can be seen that the viability of both strains at low pH is quite high This is similar to Ana Carolina Ritter et al.'s study on the viability and growth of Bacillus spp under acidic conditions In the medium Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 36 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an with pH 3, the viability of strain FTC01 reached 96% Not only that, when tested at pH 2, it has shown the ability to survive up to 90% (Ritter et al., 2018) 4.5 Effect of different carbon sources L1.5 G9.4 Optical density at 650nm 0.7 0.6 0.5 0.4 0.3 0.2 0.1 D-Gluscose Lactose Fructose Dextrin Without Carbon source Fig Effect of different carbon sources to optical absorbance The carbon sources supplied to the culture medium had a significant impact on the capacity of the two experimental bacterial strains to grow and break down cholesterol When the lactose carbon source was provided, both bacteria L1.5 and G9.4 grew slowly Three carbon sources, Lactose, Fructose, and Dextrin, inhibited the growth of strain L1.5 more than the remaining carbon sources in the experiment The optical density of microorganisms in the medium supplied with D-Glucose was 1.5 times higher than in the medium added with Dextrin When comparing the growth ability of bacteria in the medium containing Lactose to the culture in the medium containing D-Glucose, it was about 33% less than the culture in the medium containing D-Glucose Furthermore, both strains were successful in the medium without the addition of any carbon source This is explained by the activation of O2, NADPH, and H+ in the process of breaking down cholesterol, resulting in NADPH as an energy source for cell activity and development Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 37 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an (Masoud et al., 2014) When employing cholesterol as the major carbon source during the growth of three strains (GMK01, GMK02, and GMK03), this was also mentioned in Khiralla's study (Khiralla, 2015) The results of this experiment are similar to that of Yehia et al., who discovered that glucose is a useful source of carbon for Enterococcus hirae development in their research of the capacity to create extracellular cholesteroldegrading enzymes (Yehia et al., 2015) And it's vice versa, in the study of Amutha and Kokila on the cholesterol-degrading ability of Bacillus cereus strain KAVK4 isolated from butter, it was found that Fructose was the best carbon source for the growth of this bacterium (Amutha and Kokila, 2016) 4.6 Effect of different nitrogen sources Optical density at 650nm L1.5 G9.4 0.7 0.6 0.5 0.4 0.3 0.2 0.1 NaNO3 KNO3 NH4Cl NH4NO3 Nitrogen source Fig Effect of different nitrogen sources to optical absorbance Nitrogen sources support rapid growth and high cell yields of bacteria As a control, NH4NO3 was employed as a nitrogen supply in this experiment The capacity of the two bacteria strains to develop at different nitrogen sources was not significantly different When cultivated in a medium enriched with a nitrogen source, NaNO3, there were some variations in strain L1.5 The medium utilizing KNO3 as a nutrition source has an OD650 value of 0.64, whereas the Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 38 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an bacteria have the lowest OD650 value When working with the bacterium strain G9.4, the situation is radically different When cultivated in a medium enriched with NaNO3 nitrogen source, this strain showed the highest growth and development ability, however, when KNO3 was added to the medium, the strain showed the lowest performance (with an OD650 value of 0.493) The experimental results are comparable to the findings of C Tays et al., who investigated the impact of combining carbon and nitrogen sources to maximize the development of heterotrophic proteobacteria According to this study, culture media treated with ammonium produced more total biomass at OD540 than medium supplemented with nitrate (Tays et al., 2018) In another study in 2013 by Rostkowski et al., they also found that the Methylocystis parvus OBBP strain, if supplemented with ammonium as a nitrogen source, produced the best PHB, while the Methylosinus trichosporium OB3b strain in the same experiment showed the best expression (Rostkowski et al., 2013) In contrast, when the medium was supplemented with NaNO3 in research by Abouseoud et al (2008) testing alternative nitrogen sources in biosurfactant production by Pseudomonas fluorescens, the total biomass of this strain was expressed as 3.3g/L The preceding research, as well as the experimental results on two bacteria strains, L1.5 and G9.4, demonstrated the variety of the living environment as well as the optimal growth conditions of the strains in nature (Abouseoud et al., 2008) Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 39 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 4.7 Effect of different incubation time Optical density at 650nm 0.9 0.8 0.7 0.6 0.5 L1.5 0.4 G9.4 0.3 0.2 0.1 24h 48h 72h Incubation time Fig Effect of different incubation time to optical absorbance In general, microorganisms go through four phases in their life cycle: the lag phase, the log phase, the stationary phase, and the death phase (Wang et al., 2015) In the stationary phase, these microbes' growth rate reaches a maximum value (Schaechter, 2015) As a result, the last stationary phase is the optimal period for obtaining the greatest biomass for fermentation processes Therefore as reason, when the microbial population enters the death phase, the lower bacterial density will have an impact on the targeted yield when fermenting microbial strains Two strains, L1.5 and G9.4, were cultured for 24, 48, and 72 hours in this experiment to determine the best culture time Figure shows that both strains grow best when cultured for 48 hours with an OD 650 of 0.937 (L1.5) and 0.858 (G9.4), respectively Growth and development were both slow at 72 hours This might be owing to the fact that the bacterial population has entered the death phase, waste products in the culture medium have increased, and the nutrition source has decreased due to the preceding phase's increasing density Marajan et al reported that B.tequilensis strain was cultured for 48 hours of surface tension in their investigation of the influence of culture conditions on Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 40 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an the surface tension of biosurfactants generated by Bacillus spp The surface tension was lowered from 40 to 32 mN/m (Marajan et al., 2018) In contrast, when Chikere et al observed microbial strains isolated from soil, the density of Bacillus strains was found to be high (around 3.4x104 CFU/g) when the culture duration reached days (72h) (Chikere and Udochukwu, 2014) 4.8 Tolerance to bile salt Two tests for bile salt tolerance and acid resistance are of particular relevance in the research of bacteria that are advantageous to both human and animal life (Hotel and Cordoba, 2001) Bacterial strains were grown in LB medium using 0.3% ox bile salt (Sigma) and a control flask without bile salts Culture flasks are incubated at 37oC for 24h, after which the viability of bacterial strains is determined by dilution and culture on an agar plate, and the number of colonies is reported Fig Tolerance to bile salt of strains Both strains were sensitive to bile salts This was confirmed by the strain's viability and comparability when cultivated in the absence of bile salts Only 25.77% of the total bacteria of strain L1.5 survived after hours of cultivation Meanwhile, strain G9.4 only received 22.89% of the vote The results of this experiment are comparable to the findings of Lee et al., who discovered that in the presence of bile salt, pH 2.5, cell density reduced by more than 60% (from 3.18 log CFU/mL to 1.26 log CFU/mL) (Lee et al., 2015) The density of the microbial population was likewise lowered by 2.73 log CFU/mL when Jeon et al investigated the strain B subtilis P223 isolated from kimchi (Jeon et al., 2017) Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 41 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an V CONCLUSION AND SUGGESTION 5.1 Conclusion During the screening phase, the strains with the greatest ability to degrade cholesterol were chosen We chose two strains, L1.5 and G9.4, that have a high resolving power and a cholesterol degradation efficiency of 54.63 percent and 55.75 percent, respectively Through the biochemical test, two strains are both gram-positive, have endospore, are positive for the VP test and catalase test, and are negative for the methyl red reaction Furthermore, when combined with MALDI TOF, strain L1.5 was identified as Bacillus amyloliquefaciens, whereas strain G9.4 was recognized as Bacillus flexus L1.5 strain best develops at pH = 7, D-glucose, KNO3, culture time is 48 hours When strain G9.4 is cultured for 48 hours in the appropriate culture medium at pH = 7, it grows well when no carbon source is added to the medium and the nitrogen source is NaNO3 Both strains are bile salt sensitive and acid tolerant 5.2 Suggestion The research results are still positive That's this study was recommended to continue to conduct the experiment in the condition of liquid fermentation with a large capacity to observe more clearly the cholesterol-degrading ability of these two strains Therefore, extract CHO enzyme from culture solution to apply for further studies Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 42 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an REFERENCES Abouseoud M., Maachi R., Amrane A., Boudergua S and Nabi A (2008) Evaluation of different carbon and nitrogen sources in production of biosurfactant by Pseudomonas fluorescens Desalination Vol 223 pp 143-151 Alvarez, Héctor (2010) Biology of Rhodococcus Springer Science & Business Media pp 231–256 Aguiar P.M., Brito G.d.C., Correr C.J., Lyra Junior D.P and Storpirtis S (2014) Exploring the quality of systematic reviews on pharmacist interventions in patients with diabetes: an overview Annals of Pharmacotherapy Vol 48 pp 887-896 Amutha K and Kokila V (2016) CHOLESTEROL-LOWERING PROPERTY OF BACILLUS CEREUS STRAIN KAVK4 ISOLATED FROM BUTTER Asian Journal of Pharmaceutical and Clinical Research Vol pp 227-229 Anderson R.G (2003) Joe Goldstein and Mike Brown: from cholesterol homeostasis to new paradigms in membrane biology Trends in cell biology Vol 13 pp 534-539 Ballantyne CM, ed Therapeutic targeting of high-density lipoprotein metabolism In: Clinical Lipidology: A Companion to Braunwald's Heart Disease 2nd ed Philadelphia, Pa.: Saunders Elsevier; 2015 Baron, S (1996) Medical microbiology Bergey D.H (1994) Bergey's manual of determinative bacteriology: Lippincott Williams & Wilkins Berberich, A J., & Hegele, R A (2019) The complex molecular genetics of familial hypercholesterolaemia Nature reviews Cardiology, 16(1), 9-20 Chater, K F (1984) Morphological and physiological differentiation in Streptomyces Microbial development, 89-115 Chikere C and Udochukwu U (2014) Effect of growth media and incubation time on the culturability of soil bacteria IOSR Journal of Pharmacy and Biological Science Vol pp 6-9 Cleeman, J., S Grundy, D Becker and L Clark (2001) "Expert panel on detection, evaluation and treatment of high blood cholesterol, in adults Executive summary of the third report of the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP III)." Jama 285(19): 2486-2497 Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 43 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Craig M., Yarrarapu S.N.S and Dimri M (2022) Biochemistry, Cholesterol In StatPearls Treasure Island (FL) Dương Nhật Linh, Lê Thị Anh Thiện, Phạm Trần Phương Dung, Phạm Thị Minh Trang, Nguyễn Văn Minh, Trần Cát Đông (2014) Sàng lọc vi khuẩn latic có hoạt tính giảm cholesterol Tạp chí sinh học, 36 (1se) 4753 El-Naggar, N E A., El-Shweihy, N M., & El-Ewasy, S M (2016) Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase-producing Streptomyces cavourensis strain NEAE-42 BMC microbiology, 16(1), 1-20 Eyssen, H J., G G Parmentier, F C Compernolle, G de Pauw and M Piessens‐Denef (1973) "Biohydrogenation of sterols by Eubacterium ATCC 21,408—nova species." European journal of biochemistry 36(2): 411-421 Gilliland S., Nelson C and Maxwell C (1985) Assimilation of cholesterol by Lactobacillus acidophilus Applied and environmental microbiology Vol 49 pp 377-381 Gordon, T., W P Castelli, M C Hjortland, W B Kannel and T R Dawber (1977) "High-density lipoprotein as a protective factor against coronary heart disease: the Framingham Study." The American journal of medicine 62(5): 707-714 Guan, N., & Liu, L (2020) Microbial response to acid stress: mechanisms and applications Applied microbiology and biotechnology, 104(1), 51-65 Guan, X., Xu, Q., Zheng, Y., Qian, L., & Lin, B (2017) Screening and characterization of lactic acid bacterial strains that produce fermented milk and reduce cholesterol levels brazilian journal of microbiology, 48, 730-739 Hasdianty A., Suhaila Y.N., Hazwan A.H., Maniyam M.N., Fadzli A.M and Ibrahim A (2020) Inferring the evolutionary relationship of 23 Malaysian Rhodococcus isolates with potential as cholesterol degrading bacteria Biocatalysis and Agricultural Biotechnology Vol 30 pp 101840 He Q., Li J., Ma Y., Chen Q and Chen G (2021) Probiotic Potential and Cholesterol-Lowering Capabilities of Bacterial Strains Isolated from Pericarpium Citri Reticulatae ‗Chachiensis‘ Microorganisms Vol pp 1224 Hevonoja, T., M O Pentikäinen, M T Hyvönen, P T Kovanen and M AlaKorpela (2000) "Structure of low-density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL." Biochimica et Biophysica Acta (BBA)-Molecular and cell biology of lipids 1488(3): 189-210 Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 44 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Hotel A.C.P and Cordoba A (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria Prevention Vol pp 1-10 Hucker, G J., & Conn, H J (1923) Methods of Gram staining Jeon H.-L., Lee N.-K., Yang S.-J., Kim W.-S and Paik H.-D (2017) Probiotic characterization of Bacillus subtilis P223 isolated from kimchi Food Science and Biotechnology Vol 26 pp 1641-1648 Jha R., Das R., Oak S and Mishra P (2020) Probiotics (direct-fed microbials) in poultry nutrition and their effects on nutrient utilization, growth and laying performance, and gut health: a systematic review Animals Vol 10 pp 1863 Jose J (2016) Statins and its hepatic effects: Newer data, implications, and changing recommendations Journal of pharmacy & bioallied sciences Vol pp 23 Kämpfer P (2006) The Family Streptomycetaceae, Part I: Taxonomy In The Prokaryotes: Volume 3: Archaea Bacteria: Firmicutes, Actinomycetes (Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H and Stackebrandt, E eds) New York, NY: Springer New York, pp 538-604 Khánh H.Q and Thanh P.T.L (2011) Phân lập, định danh xác định chủng Lactobacillus có tiềm probiotic từ người Tạp chí Phát triển Khoa học Cơng nghệ Vol 14 pp 62-76 Khiralla G.M (2015) Cholesterol degradation by some bacteria isolated from food Food Science and Technology Research Vol 21 pp 685-693 Kim K., Rhee C and Park H (2002) Degradation of cholesterol by Bacillus subtilis SFF34 isolated from Korean traditional fermented flatfish Letters in applied microbiology Vol 35 pp 468-472 Kokila V and Amutha K (2016) Cholesterol degrading potentiality of the bacterial isolates from Ghee International Journal of Pharmaceutical Sciences Review and Research Vol 39 pp 255-259 Lân D., Nguyễn Q and Pham T (2010) Vi sinh vật học NXB Giáo dục Việt Nam Vol pp Lashkarian, H., Raheb, J., Shahzamani, K., Shahbani, H., & Shamsara, M (2010) Extracellular cholesterol oxidase from Rhodococcus sp.: isolation and molecular characterization Iranian Biomedical Journal, 14(1-2), 49 Lee N.-K., Son S.-H., Jeon E.B., Jung G.H., Lee J.-Y and Paik H.-D (2015) The prophylactic effect of probiotic Bacillus polyfermenticus KU3 against cancer cells Journal of functional foods Vol 14 pp 513-518 Lee, N K., Kim, W S., & Paik, H D (2019) Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier Food science and biotechnology, 28(5), 1297-1305 Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 45 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Leikind, M C (1939) Bacteria, the Smallest of Living Organisms By Dr Ferdinand cohn (1872) Translated by Charles S Dolley (1881) Bulletin of the History of Medicine, 7, 49 Lin E.C., Fernandez M.L., McNamara D.J Dietary fat type and cholesterol quantity interact to affect cholesterol metabolism in guinea pigs J Nutr 1992;122:2019–2029 doi: 10.1093/jn/122.10.2019 Linton, M F., Yancey, P G., Davies, S S., Jerome, W G., Linton, E F., Song, W L., & Vickers, K C (2019) The role of lipids and lipoproteins in atherosclerosis Lnsis A (2000) Atherosclenrosis Nature Vol 407 pp 233-241 Madsen C.M., Varbo A and Nordestgaard B.G (2017) Extreme high highdensity lipoprotein cholesterol is paradoxically associated with high mortality in men and women: two prospective cohort studies Eur Heart J Vol 38 pp 2478-2486 Marajan C., Alias S., Ramasamy K and Abdul-Talib S (2018) The effect of incubation time, temperature and pH variations on the surface tension of biosurfactant produced by Bacillus spp In AIP Conference Proceedings: AIP Publishing LLC, pp 020047 Masoud R., Bizouarn T and Houée-Levin C (2014) Cholesterol: A modulator of the phagocyte NADPH oxidase activity-A cell-free study Redox biology Vol pp 16-24 Mc Namara K., Alzubaidi H and Jackson J.K (2019) Cardiovascular disease as a leading cause of death: how are pharmacists getting involved? Integrated pharmacy research & practice Vol pp Mollahosseini M., Shab-Bidar S., Rahimi M.H and Djafarian K (2017) Effect of whey protein supplementation on long and short term appetite: A metaanalysis of randomized controlled trials Clin Nutr ESPEN Vol 20 pp 34-40 Miyajima, C., Iwaki, T., Umemura, K., Ploplis, V A., & Castellino, F J (2018) Characterization of atherosclerosis formation in a murine model of type IIa human familial hypercholesterolemia BioMed Research International, 2018 National Center for Biotechnology Information (2021) PubChem Compound Summary for CID 5997, Cholesterol Retrieved October 31, 2021 from https://pubchem.ncbi.nlm.nih.gov/compound/Cholesterol Nguyễn Lân Dũng, Đinh Thúy Hằng, 2006 Phương pháp thực nghiệm dùng để định tên loài vi khuẩn, Nhà xuất giáo dục Oikonomou E., Siasos G., Tsigkou V., Bletsa E., Panoilia M.-E., Oikonomou I.N., Simanidis I., Spinou M., Papastavrou A and Kokosias G (2020) Coronary artery disease and endothelial dysfunction: novel diagnostic and Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 46 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an therapeutic approaches Current Medicinal Chemistry Vol 27 pp 10521080 Pal S., Ellis V and Dhaliwal S (2010) Effects of whey protein isolate on body composition, lipids, insulin and glucose in overweight and obese individuals British journal of nutrition Vol 104 pp 716-723 Parekh S and Desai P (2013) Isolation and characterization of extracellular cholesterol oxidase producing Microbacterium sp from waste of regional oil mill Int J Adv Life Sci Vol pp 87-92 Pereira, D I., & Gibson, G R (2002) Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut Applied and environmental microbiology, 68(9), 4689-4693 Pirahanchi, Y., Sinawe, H., & Dimri, M (2021) Biochemistry, LDL cholesterol StatPearls Publishing Pham Manh Hung (2011) Rối loạn lipid máu nguy bệnh tim mạch, Hội Tim mạch học Quốc gia Việt Nam Visit on 13/01/2021 http://vnha.org.vn/tapchi/RoiLoanLipidMau.indd.pdf Pousinho S., Morgado M., Falcão A and Alves G (2016) Pharmacist interventions in the management of type diabetes mellitus: a systematic review of randomized controlled trials Journal of managed care & specialty pharmacy Vol 22 pp 493-515 Ritter A.C., Paula A., Correa F., Veras F and Brandelli A (2018) Characterization of Bacillus subtilis available as probiotics Journal of Microbiology Research Vol pp 23-32 Rostkowski K.H., Pfluger A.R and Criddle C.S (2013) Stoichiometry and kinetics of the PHB-producing Type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP Bioresource technology Vol 132 pp 71-77 Rudel L.L and Morris M (1973) Determination of cholesterol using ophthalaldehyde Journal of Lipid Research Vol 14 pp 364-366 Ruiz, L., A Margolles and B Sánchez (2013) "Bile resistance mechanisms in Lactobacillus and Bifidobacterium." Frontiers in microbiology 4: 396 Sardessai Y and Bhosle S (2003) Isolation of an organic-solvent-tolerant cholesterol-transforming Bacillus species, BC1, from coastal sediment Marine Biotechnology Vol pp 116-118 Schaeffer, Alice B.; Fulton, MacDonald (1933) ―A Simplified Method of Staining Endospores‖ Science 77 (1990) Schaechter M (2015) A brief history of bacterial growth physiology Frontiers in microbiology Vol pp 289 Shobharani P and Halami P.M (2016) In vitro evaluation of the cholesterolreducing ability of a potential probiotic Bacillus spp Annals of Microbiology Vol 66 pp 643-651 Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 47 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an SMITH A.G and BROOKS C.J (1977) The mechanism of the isomerization of cholest-5-en-3-one to cholest-4-en-3-one by cholesterol oxidase: Portland Press Ltd Starks MA, Schmicker RH, Peterson ED, May S, Buick JE, Kudenchuk PJ, et al Association of neighborhood demographics with out-of-hospital cardiac arrest treatment and outcomes, where you live may matterexternal icon JAMA Cardiol 2017;2(10):1110–1118 Tang W.W., Kitai T and Hazen S.L (2017) Gut microbiota in cardiovascular health and disease Circulation research Vol 120 pp 1183-1196 Tays C., Guarnieri M.T., Sauvageau D and Stein L.Y (2018) Combined effects of carbon and nitrogen source to optimize growth of proteobacterial methanotrophs Frontiers in microbiology Vol pp 2239 Ta, H., Lin, B., & Palaniappan, L Vietnamese and Vietnamese-American Health Statistics, 2003-2019 Van der Geize, R., Yam, K., Heuser, T., Wilbrink, M H., Hara, H., Anderton, M C., & Eltis, L D (2007) A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages Proceedings of the National Academy of Sciences, 104(6), 1947-1952 Van Eck, M., Twisk, J., Hoekstra, M., Van Rij, B T., Van der Lans, C A., Bos, I S T., & Van Berkel, T J (2003) Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver Journal of Biological Chemistry, 278(26), 23699-23705 Virani SS, Alonso A, Aparicio HJ, Benjamin EJ, Bittencourt MS, Callaway CW, et al Heart disease and stroke statistics—2021 update: a report from the American Heart Associationexternal icon Circulation 2021;143:e254– e743 Wang, Y., Z Lu, H Wu and F Lv (2009) "Study on the antibiotic activity of microcapsule curcumin against foodborne pathogens." International journal of food microbiology 136(1): 71-74 Wali H., Rehman F.U., Umar A and Ahmed S (2019) Cholesterol degradation and production of extracellular cholesterol oxidase from Bacillus pumilus W1 and Serratia marcescens W8 BioMed research international Vol 2019 pp Wang L., Fan D., Chen W and Terentjev E.M (2015) Bacterial growth, detachment and cell size control on polyethylene terephthalate surfaces Scientific reports Vol pp 1-11 When, H D L (2016) cholesterol doesn‘t protect against heart disease NIH WHO (2016) Issue preview: Cardiovascular diseases (CVD) in Viet Nam Retrieved on 01 May 2022 at https://www.who.int/vietnam/healthtopics/cardiovascular Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn 48 C.vT.Bg.Jy.Lj.Tai lieu Luan vT.Bg.Jy.Lj van Luan an.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Stt.010.Mssv.BKD002ac.email.ninhd.vT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.LjvT.Bg.Jy.Lj.dtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn