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 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 iv LIST OF TABLES Table Components of the medium 21 Table The efficiency of cholesterol degradation 31 Table Characterization biochemical of selected strains 32 v 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 vi 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 vii 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 viii 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 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) 35 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 36 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 37 (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 38 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) 39 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 40 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) 41 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 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