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. 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.
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. ii LIST OF TABLES .v LIST OF FIGURES.v LIST OF ABBREVIATIONS. Purpose and requirements. Scientific and practical significance.
Introduction to cholesterol. An overview of Bacillus strains that could break down cholesterol. General information about Bacillus spp. Some Bacillus strains able cholesterol degrade.
Determining cholesterol concentration in microorganisms. Bacillus strains that degrade cholesterol are being studied in Vietnam and across the world. MATERIAL, AND METHODS. Materials, chemicals, and equipment.
Chemicals and equipment. Screening for cholesterol degrading microorganism. Characterization biochemical of selected strains. Identification of strains L1.
Effect of different pH. Effect of different carbon sources. Effect of different nitrogen sources. Effect of different incubation times.
Tolerance to Bile Salt. Screening for Bacillus strains capable of degrading cholesterol. Characterization biochemical of selected strains. Identification of strains L1.
Effect of different pH. Effect of different carbon sources. Effect of different nitrogen sources. Effect of different incubation time.
Tolerance to bile salt. CONCLUSION AND SUGGESTION .43 iv LIST OF TABLES Table 3. 1 Components of the medium. 1 The efficiency of cholesterol degradation.
2 Characterization biochemical of selected strains .32 v LIST OF FIGURES Fig 2. 1 Chemical structure of cholesterol. 2 Low density lipoproteins (LDL). 4 Cholesterol-rich plaque (yellow) builds up inside your arteries.
5 Reaction catalyzed by cholesterol oxidase. subtilis 168 obtained using a scanning electron microscope. 7 Process of MALDI TOF. 1 Cholesterol Standard Curve.
2 Microorganism strains capable of degrading cholesterol. 3 Biochemical test of selected strains. 4 Effect of different pH to optical absorbance. 5 Acid tolerance of L1.4 strain at pH 3.
6 Effect of different carbon sources to optical absorbance. 7 Effect of different nitrogen sources to optical absorbance. 8 Effect of different incubation time to optical absorbance. 9 Tolerance to bile salt of 2 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 (L1.4) that could use cholesterol as a source of nutrients.4 exhibited a cholesterol degradation percentage of 55.
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.
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. Many researchers have achieved impressive clinical outcomes on the relationship between patients undergoing CVD therapy and blood cholesterol decrease (Pousinho et al.
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. Exogenous cholesterol is created by the consumption of animal fat- containing 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 1 for one-third of all ischemic heart disease worldwide (Dương Nhật Linh et al.
Currently, medical centers frequently utilize statins, which block HMG- CoA (β-Hydroxy β-methylglutaryl-CoA) reductase, do not produce cholesterol in the liver, and lower blood cholesterol. However, this drug can cause liver dysfunction, elevate SGOT (serum glutamic-oxaloacetic transaminase) or SGPT (serum glutamic pyruvic transaminase) liver enzymes, and lead to hepatic necrosis in individuals. When taking statin drugs, patients may experience flatulence, indigestion, diarrhea, constipation, abdominal pain, and loss of appetite (Jose, 2016). Nowadays, the use of microbes in the treatment of human and animal illnesses is frequently suggested.
A molecular relationship between gut microbiota and the severity of myocardial infarction has recently been revealed. In atherosclerosis research, fibrous areas harboring bacterial DNA recovered quicker than equivalent regions without bacteria (Tang et al. A cholesterol oxidase (cholesterol: oxygen oxidoreductase, EC 1.6), an enzyme that catalyzes the oxidation of cholesterol to 4-cholesten-3-one while also reducing oxygen to hydrogen peroxide, is known to degrade cholesterol in bacteria (SMITH and BROOKS, 1977). Many studies have demonstrated that microbes can decrease cholesterol levels in recent years.
Eyssen et al.,(1973) identified bacteria capable of degrading cholesterol from the cecum of rats. For growth, these organisms require free or esterified cholesterol. Another study by Duong Nhat Linh et al., (2014) examined 32 strains of lactic acid bacteria isolated from breast milk, newborn meconium, pickled vegetables, and naturally fermented yogurt for the production of BSH enzyme, which eliminates cholesterol via surface binding. Kokila and Amutha (2016) of Vels University (India) discovered the cholesterol-degrading bacterium Bacillus.
subtilis KAVK3 from Ghee avocado. 2 Despite the fact that much research on the effect of Bacillus on cholesterol degradation has been conducted across the world. However, research in this field is relatively restricted in Vietnam. The purpose of this study was to: ―Biochemical characterization of cholesterol-degrading Bacillus strains.
Purpose and requirements 1. Purpose Evaluating the capacity to break down cholesterol under various growth conditions, to apply it to the manufacturing of probiotic-containing products. Requirements Determining the growth ability of selected bacterial strains on cholesterol-containing media under different culture conditions. Scientific and practical significance 1.
Scientific significance The study findings serve as a foundation for future studies on bacillus bacteria and cholesterol reduction, as well as research and development of probiotics derived from Bacillus strains for use in the treatment of CVDs. Practical significance The study results will add to our understanding of Bacillus strains that may break down cholesterol. To assess how the culture environment affects the growth and development of the experimental strains. The selection and evaluation of microbial strains' cholesterol-degrading capacity help to enhance helpful microbial items for biological therapeutic development.
As a result, people's quality of life improves while treatment efficacy improves. Introduction to cholesterol Cholesterol is a precursor of steroid hormones, bile acids, and vitamin D and may be found in cell membranes. We make the majority of our cholesterol, but some come from our diet. Cholesterol synthesis is a pathway that occurs in the smooth endoplasmic reticulum of a cell (Craig et al.
It begins with acetyl-CoA molecules being joined together by the enzyme acetyl coaxial transfer case, yielding a four-carbon molecule known as acetylcholine. Cholesterol has been linked to atherosclerosis, one of the leading causes of cardiovascular disease (Lnsis, 2000). Atherosclerosis is caused by plaque buildup on the interior walls of arteries, which is made up of fat, cholesterol, calcium, and other chemicals present in the blood. It's a complicated process including oxidized low-density lipoproteins (LDL) and a persistent inflammatory reaction on artery walls.This causes a pathologic build-up of LDL in blood vessels and the production of atherosclerotic plaques, resulting in blood vessel constriction (Oikonomou et al.
Our bodies manufacture lipoproteins to dilute cholesterol and other lipids in our circulation that are water-insoluble. Cholesterol has two main types of lipoproteins: Low-density lipoprotein cholesterol (LDL cholesterol) is what makes up cholesterol, while high-density lipoprotein cholesterol (HDL cholesterol) is what transports cholesterol to and from cells in the body. A blood test may be used to determine the levels of each kind of cholesterol in the blood (Pham Manh Hung, 2011). 1 Chemical structure of cholesterol (Source: https://www.net/publication/286928214_Effects_of_static_vs_non- static_in_vitro_techniques_on_lipid_penetration_into_silicone_hydrogel_contact_lenses ) 2.
Low-density lipoprotein cholesterol (LDL cholesterol) LDL cholesterol, also known as low-density lipoprotein cholesterol, is a kind of fat that circulates in the bloodstream, transporting cholesterol across the body to where it is needed for cell repair and depositing it inside artery walls. Because cholesterol and triglycerides are insoluble in water, they must be bound to proteins circulating in the hydrophilic blood (Pirahanchi et al. 2 Low density lipoproteins (LDL) (Source: https://www.net/health/LDL-Cholesterol-and-Heart- Disease.aspx ) 5 LDL is formed from LDL secondary lipase with modification and peripheral tissue and the liver. It is endocytosed by target sold with LDL receptors and degraded relieving cholesterol which decrees has further uptake of cholesterol is cholesterol problem delivered to peripheral tissues, It is commonly referred to as bad cholesterol.
LDL cholesterol transports the majority of the cholesterol in the body, accounting for 60-70 % of total cholesterol in the blood. When this concentration in the blood rises significantly, there is a danger of fat deposition in vessel walls (particularly in the heart and lungs), which can lead to atherosclerosis (When,2016). However, LDL is also known as bad cholesterol, which affects a lot of health.