VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: FERMENTATION OF GINSENG EXTRACTS BY PROBIOTIC BACTERIA AND THEIR ANTIMICROBIAL AND ANTI-OXIDANT ACTIVITY HANOI, 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: FERMENTATION OF GINSENG EXTRACTS BY PROBIOTIC BACTERIA AND THEIR ANTIMICROBIAL AND ANTI-OXIDANT ACTIVITY Student : Le Minh Vy Supervisor : Dr. Bui Thi Thu Huong Dr. Le Thi Hoang Yen Dr. Vu Duy Nhan Student code : 610691 Class : K61CNSHE Location : Institute of Microbiology and Biotechnology, VNUA HANOI, 2021 DECLARATION I hereby declare that this paper was my own work.
All results and data in this title were absolutely honest and have not been submitted before to any institution for assessment purposes. All sources used in this paper were cited in references. Student Le Minh Vy i ACKNOWLEDGEMENTS I would like to express my respect and deep gratitude to Dr. Bui Thi Thu Huong, Dr.
Le Thi Hoang Yen and Dr. Vu Duy Nhan, for giving me the opportunity to carry out this work, and their huge efforts, enthusiasm, and support throughout the duration of the undergraduate thesis. I would like to express sincere gratitude to all the staff of the Institute of Microbiology and Biotechnology who were enthusiastic to help me through the project. Finally, I would like to thank my family, those with us and those sadly departed.
You provided fantastic support and inspiration throughout the duration of this undergraduate thesis, and without you all, this would never have been possible. Student Le Minh Vy ii CONTENTS DECLARATION .iii LIST OF TABLE. v LIST OF FIGURE. vi PART I: INTRODUCTION.
Objectives and requirements. 1 PART II: OVERVIEW. Introduction of ginseng. Ingredients and uses.
Overview of fermented ginseng. Methods of ginsenoside metabolism. Mechanism of the fermentation method. 9 PART III: MATERIALS, CONTENT AND METHOD OF RESEARCH.
Materials and equipments. Experimental equipment and tools. Methods of optimizing culture conditions. Analysis methods of saponin content.
13 iii PART IV: RESULTS AND DISCUSSION. Check for the survival of bacteria. Evaluation of ginsenoside metabolism of strain NL812 in ginseng medium. Optimizing culture conditions.
Effect of temperature. Effect of the initial pH value. Effect of carbon sources. Effect of nitrogen sources.
Effect of fermented times. Analyze the transformation of ginseng by TLC and HPLC method. CONCLUSIONS AND RECOMMENDATIONS. 32 iv LIST OF TABLE Table 4.
Effect of temperature. Effect of the initial pH value. Effect of carbon sources. Effect of nitrogen sources.29 v LIST OF FIGURE Fig 2.
Structure of saponins. Mechanism of β - glucosidase. β-glucosidase in the hydrolysis. Graph of standard Yonagenin.
Colony of bacterial NL812. Saponin of ginseng medium. Effect of temperature. Effect of the initial pH value.
Effect of carbon sources. Effect of nitrogen sources. Effect of fermented times. TLC of ginseng extract before fermentation.
TLC of ginseng extract after fementation. Chromatogram of the C-K standard. Chromatogram of the Rg3 standard. Chromatogram of the TTB.
The chromatogram of the TTB sample was fermented by NL812 .29 vi PART I: INTRODUCTION 1. Preface In recent years, the application of bacteria in biotransformation to produce pharmacologically valuable secondary ingredients is being studied extensively in medicinal plants. One of the most valuable and researched medicinal plants is ginseng. Panax ginseng has been used as a folk medicine for healing and maintaining health for thousand years in Asian countries such as Korea, China and Japan.
Ginseng saponins, known as ginsenosides, have been considered as the main ingredients of ginseng's pharmacological effects. Nowadays, more than 150 ginsenoside derivatives have been found from the roots, leaves, berries, and buds of ginseng. The pharmacological effects of ginsenosides depend on the types or location of sugars attach to dammarane or oleanane with aglycones molecule. The main ginsenosides in the roots of ginseng are ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1, which account for 80-90% of the total number of ginsenosides (Jitendra et al.
Other methods, such as acid hydrolysis, separate alkali , and microbial and enzyme decomposition to produce low-weight molecular ginsenosides Rg3, F2, Rh2 and compound K from major ginsenosides, so that the human body metabolize and absorb easier. Microbiological or enzyme hydrolysis methods are more prevalent than conventional chemical methods because the reaction occurs in basic conditions, reaction steps are simple and intensive. However, some research in produce low- weight molecular ginsenoside by enzyme have been limited by low productivity, the complexity of enzyme method, or food safety problem. Therefore, we conduct to research the subject: “Fermentation of ginseng extracts by probiotic bacteria and their antimicrobial and anti oxidant activity” 1.
Objectives and requirements 1. Objectives • Determining the suitable concentration of ginseng for fermentation • Establishing conditions that affect ginsenoside metabolism 1. Requirements • Researching and optimizing the conditions affect the ability of metabolize ginsenoside from bacteria NL812 • Determination the ginsenoside metabolism of β-glucosidase enzyme by thin- layer chromatography (TLC) analysis and high-pressure liquid chromatography (HPLC). 1 PART II: OVERVIEW 2.
Introduction of ginseng 2. Ginseng Ginseng (Panax ginseng) is a species of flowering plant in the Araliaceae family. Ginseng is the root of plants in the genus Panax, such as Korean ginseng (P. ginseng), South China ginseng (P.
notoginseng), and American ginseng (P. quinquefolius), typically characterized by the presence of ginsenosides and gintonin. Ginseng is found in cooler climates – Korean Peninsula, Northeast China, and Russian Far East, Canada and the United States, although some species grow in warm regions – South China ginseng being native to Southwest China and Vietnam. Although ginseng has been used in traditional medicine over centuries, modern clinical research is inconclusive about its medical effectiveness.
There is no substantial evidence that ginseng is effective for treating any medical condition, and its use has not been approved by the US Food and Drug Administration (FDA) as a prescription drug. Although ginseng is commonly sold as a dietary supplement, inconsistent manufacturing practices for supplements have led to analyses showing that ginseng products may be contaminated with toxic metals or unrelated filler compounds, and its excessive use may have adverse effects or untoward interactions with prescription drugs. Nowadays, modern medicine has many studies that confirm pharmacological effects of ginseng as boost energy, improve memory, reduce stress, effects on the immune system against inflammation, protect cells against aging, increase the body's resistance. Moreover, many new effects of ginseng were discovered, such as anti- oxidants, anti-cancer.
Meanwhile, in Vietnam, people discovered many kinds of ginseng: Ngoc Linh ginseng is also known as Vietnamese ginseng, grows in the mountainous districts of Ngoc Linh, Quang Nam province. As one of the 5 most valuable ginseng in the world. It is known to many people for its use in stopping bleeding, healing wounds, tonic to help restore health quickly, dispel fatigue. Especially Ngoc Linh ginseng also has anti-aging and anti-cancer effects.
2 Bo Chinh ginseng is also known as Tho Hao ginseng (Abelmoschus sagittifolius) is a wild ginseng species, mainly distributed in the southern central provinces and Tay Nguyen such as Phu Yen, Binh Dinh, Gia Lai. It is used to treat cough, fever, asthenic health or in general weakness, thinness, or lose weight. This ginseng is also used as a tonic for health, regulating menstruation, treating pneumonia or leukemia, pimples, scabies. Stone ginseng (Myxopyrum smilacifolium Bl) belongs to the Oleaceae, also has another name as Nhuong Le Kim Cang.
It has been recently discovered in arid mountainous regions in the Northwest provinces. It has a good effect on restoring health and strengthening the body, increasing vitality and fostering tendons, good for kidney and body weakness treatment. This is also an extremely good herb for people with heart disease. Forest areca ginseng (Curculigo orchioides Gaertn) is a line of ginseng that grows naturally in the mountains of high mountainous provinces in our country such as Dien Bien, Ha Giang, Cao Bang, Bac Kan.
In addition, forest areca ginseng is also found in some other countries such as Thailand, Malaysia, India or the Philippines. Forest areca ginseng is used as a medicinal ingredient in Oriental medicine with the name 'mao'. The drug has spicy taste attributable to the kidney, treatment of diarrhea, rheumatism, impotence, weak physiology, decline sexual function in men. Panax pseudoginseng is found in the mountainous provinces of Vietnam such as Ha Giang, Lao Cai, and Cao Bang.
It is very rare in Vietnam because this tree grows only in mountainous areas with an altitude of 1,500m or more, which is suitable for cold climates. Ginseng root is used as an ingredient in medicine, the root contains a lot of rare and healthy pharmaceuticals. In addition, the flowers and buds are also used to treat cardiovascular disease and blood pressure. Panax pseudoginseng is usually harvested after 5 to 7 years, people usually harvest in November every year after the tree has reached the age of 5-years or more.
Analyzing the composition in the Panax pseudoginseng, the scientists discovered that there are many rare nutrients and pharmaceuticals, including amino 3 acids, sterols, sugars, Fe, Ca, especially 2 substances Saponin: Arasaponin A, Arasaponin B. Ingredients and uses Studies have shown that ginseng has many uses such as: elevate resistance, regulating the nervous system, anti-convulsions, anti-aging, regulating blood circulation. Nowadays, scientists discovered that ginseng has anti-cancer effects, inhibits metastasis and regenerates cancer cells, promotes cell differentiation. The chemical composition of ginseng includes: ginsenoside, oligosaccharides, polysaccharides, amino acids, proteins, oils and fatty acids, etc.
In which the active ingredient is mainly ginsenoside. They are divided into 2 groups: Protopanaxadiol-PD like Rb1, Rb2, Rc, Rd and Protopanaxatriol PT group like Re, Rf, Rg1, Rg2. In which, Rb1, Rd, Re account for most content in ginseng. In contrast, the ginsenoside Rg1, Rg3, Rh1, Rh2 in natural conditions are very small, but these ginsenosides are the main components that make up the pharmacological properties of ginseng: Ro: It has the effect of breaking down alcohol, preventing hepatitis and restoring bad breath Rb1: It could inhibit the central nervous system so that pain is relieved Rb2: Preventing diabetes restriction, liver sclerosis, and accelerate liver absorption Rc: Reduce the pain, speeds up protein absorption Rg1, Rg2, Rg3: Anti-fatigue, reduce stress, restore memory, prevent cancer cells and protect the liver.
Rh1, Rh2: Inhibits cancer cells, protects the liver, prevents platelet binding 4 Fig 2. Structure of saponins In recent years, researchers have focused on studying some rare ginsenosides, such as Panaxadiol Saponifier Rg3, Rh2, CK, etc., which have pharmacological activities such as anti-cancer, anti-inflammatory, elevate immunity,. However, the content of these rare ginsenosides in ginseng is very small, there are types ginsenoside in ginseng that naturally does not exist, only exist in red ginseng (Park et al., 2014, Lee et al., 2016, Sun et al. More than 150 ginsenoside derivatives have been identified from the roots, leaves, berries, and buds of P.
ginseng and other types of ginseng. The pharmacological effects of ginsenosides often depend on the type of sugars locate on aglycones (dammarane or oleanane). The main ginsenosides in the roots of P. ginseng are ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1, which account for 80-90% of the total number of ginsenosides.
However, these naturally occurring major ginsenosides are poorly absorbed through the human intestinal because of their molecular size, low solubility and poor permeability on cell membranes. Meanwhile, the low-weight molecular ginsenosides Rg3, F2, Rh2 hydrolyzed from the above main ginsenosides can be easily metabolized and absorbed in the human body. 5 In addition, small ginsenosides such as Rg3, Rh2 are easy to solube and absorb through human’s intestine, especially Rg3, although it accounts for a low rate, only approximately 4. (by 1/6 Rb1), but they have pharmacological activities such as anti-cancer, anti-inflammatory, increased immunity.
etc (Park et al., 2014, Lee et al., 2016, Sun et al. In this study, we investigated saponin metabolism and increase.